CN114736929A - Composition, method and application for generating recombinant baculovirus in insect cells - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly discloses a composition, a method and application for generating recombinant baculovirus in insect cells. The recombinant baculovirus prepared by the composition provided by the invention can be obtained only by one-step recombination in host insect cells without screening and the restriction of a single recombination site, and the purity of the prepared recombinant baculovirus is higher.

Description

Composition, method and application for generating recombinant baculovirus in insect cells

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a composition, a method and application for generating recombinant baculovirus in insect cells.

Background

Baculoviruses are a class of circular double-stranded DNA viruses with an envelope. Currently, the most studied baculovirus strain is autographa californica (autographa californica) polynuclear polyhedrosis virus (MNPV), abbreviated as AcMNPV. The baculovirus expression system (BEVS) is a eukaryotic expression system that uses insect baculovirus vectors to infect host insect cells to produce foreign proteins.

Kitts et al have proposed BacPAK baculovirus expression system, this system inserts lacZ gene into the polyhedrosis virus locus position, have constructed a kind of replication defective virus linearized after Bsu 36I enzyme digestion through introducing a Bsu 36I enzyme digestion site in orf1629 and orf603 gene site respectively, virus after linearization, because of deleting essential gene orf1629, can't produce the virus with infectious activity even if the self-connection, will rescue the recombinant DNA transfer vector and linearized virus and co-transfect host insect cell, get the recombinant baculovirus after homologous recombination. However, since the Bsu 36I enzyme cannot cut 100% of the viral DNA for linearization, recombinant viruses still require plaque screening, which is time and labor consuming.

Subsequently, Lee et al have proposed a Bac-to-Bac baculovirus expression system that introduces a Bac artificial chromosomal element containing a mini-F replicon and a Tn7 transposition site at the polyhedric gene site of AcMNPV, allowing baculovirus DNA to replicate in bacteria while allowing insertion of foreign genes into the viral genome by Tn7 transposition to obtain recombinant baculoviruses. Although the system does not need to carry out plaque screening, transposition recombination screening in bacteria is needed, and the system is time-consuming and labor-consuming; and the recombinant baculovirus obtained by the system has unstable phenomenon in continuous passage.

In 2003, zhao et al have proposed a flash-BAC system that inactivates the essential gene orf1629 while introducing the mini-F replicon into the AcMNPV genome, and baculoviruses that inactivate the essential gene orf1629 cannot produce infectious virus unless homologous recombination occurs with the rescue recombinant DNA transfer vector. The system combines the advantages of BacPAK and Bac-to-Bac, and can obtain the recombinant baculovirus only by one-step recombination in the host insect cell without screening. However, since orf1629 is a trans-element protein involved in replication, the viral DNA in which homologous recombination has occurred will provide the function of orf1629, and since the viral DNA in which homologous recombination has not occurred will also be packaged, resulting in impure recombinant baculovirus finally obtained.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a composition, a method and an application for producing recombinant baculovirus in insect cells, and aims to solve the problems that viral DNA which is not subjected to homologous recombination in the process of producing the recombinant baculovirus by using a flash-BAC system is packaged, and the viral DNA which is subjected to homologous recombination provides the function of orf1629, so that the recombinant baculovirus is impure.

To achieve the above object, the present invention provides a composition for producing a recombinant baculovirus in an insect cell, comprising a packaging-defective baculovirus plasmid that deletes a CNE sequence and/or a NAE sequence in a baculovirus genome, and a first rescue recombinant DNA comprising a first insertion sequence comprising a first functional fragment and a first complementing sequence that is at least one of the sequences deleted from the packaging-defective baculovirus plasmid and a first homology arm located on both sides of the first insertion sequence, the composition being capable of undergoing homologous recombination in an insect cell to produce a recombinant baculovirus.

Preferably, the first functional fragment is a cap gene expression cassette of AAV and a rep gene expression cassette of AAV.

Preferably, said first insertion sequence comprises, in order from 5 'to 3', said cap gene expression cassette, said first complementing sequence and said rep gene expression cassette.

Preferably, the first insertion sequence comprises, in order from 5 'to 3', the rep gene expression cassette, the first complementing sequence and the cap gene expression cassette.

Preferably, the first complementing sequence is located between the cap gene expression cassette and the rep gene expression cassette, and two ends of the first complementing sequence are respectively close to the starting end of the cap gene expression cassette and the starting end of the rep gene expression cassette.

Preferably, the packaging-defective baculovirus plasmid is a recombinant bacmid comprising an AAV ITR core expression element with a foreign gene.

Preferably, the recombinant bacmid is obtained by baculovirus transfer vector mediated transposition of Tn 7.

Preferably, the first functional fragment is an AAV ITR core expression element with a foreign gene.

Preferably, the packaging-defective baculovirus plasmid is a recombinant bacmid comprising an AAV cap gene expression cassette and an AAV rep gene expression cassette.

Preferably, the recombinant bacmid is obtained by baculovirus transfer vector mediated transposition of Tn 7.

Preferably, the first functional fragment is a reporter gene.

Preferably, the first functional fragment is a nucleotide sequence encoding a therapeutic gene product.

Preferably, the composition further comprises a second rescue recombinant DNA missing a CNE sequence and a NAE sequence in the baculovirus genome from the packaging-defective baculovirus plasmid, the second rescue recombinant DNA comprising a second insertion comprising a second anaplerotic sequence, the first anaplerotic sequence being a CNE sequence or a NAE sequence, and a second homology arm flanking the second insertion, the second anaplerotic sequence being a sequence other than the first anaplerotic sequence of the CNE sequence and the NAE sequence.

Preferably, the first functional fragment is a cap gene expression cassette of AAV and a rep gene expression cassette of AAV, and the second insertion sequence further comprises a second functional fragment which is an AAV ITR core expression element with a foreign gene.

According to another aspect of the present invention there is provided the use of any one of the compositions described above for the preparation of recombinant baculovirus and/or recombinant adeno-associated virus in insect cells.

According to another aspect of the present invention, there is provided an insect cell comprising any one of the compositions described above.

According to another aspect of the present invention there is provided a method of growing or producing a recombinant baculovirus in vitro comprising co-transfecting an insect cell with any of the compositions described above and culturing the insect cell.

According to another aspect of the present invention, there is provided a method of growing or producing a recombinant adeno-associated virus in vitro, comprising co-transfecting an insect cell with any of the compositions described above and culturing the insect cell.

Generally, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:

the invention is improved on the basis of a flash-BAC system, and the packaging defect type baculovirus plasmid is constructed, lacks the CNE sequence and/or the NAE sequence in the baculovirus genome, and can not normally package baculovirus unless homologous recombination with rescue recombinant DNA occurs. The system can obtain recombinant baculovirus only by one-step recombination in host insect cells without screening; meanwhile, the insertion sequence can be inserted into any gene locus of the baculovirus genome without the limitation of an insertion site, can be inserted into a target site according to needs, can realize the expression of multiple foreign proteins from a single baculovirus, and has stronger flexibility. In addition, when a recombinant baculovirus is prepared using this system, only viral DNA that undergoes homologous recombination is packaged into a recombinant baculovirus, and viral DNA that does not undergo homologous recombination is not packaged, so that the recombinant baculovirus finally obtained is purer.

Drawings

FIG. 1 is a schematic diagram of a first homologous recombination expression cassette of a targeted CNE sequence constructed in example 1 of the present invention.

FIG. 2 is a schematic diagram of a second homologous recombination expression cassette of a targeted NAE sequence constructed in example 1 of the present invention.

FIG. 3 is a schematic diagram of a third homologous recombination expression cassette of a targeted NAE sequence constructed in example 1 of the present invention.

FIG. 4 is a diagram showing a recombinant DNA fragment Ac96-CNE-GFP constructed in example 2 of the present invention, which has an insertion site at orf 96.

FIG. 5 is a diagram showing a recombinant DNA fragment Ac96-NAE-mcherry having an insertion site at orf96 constructed in example 2 of the present invention.

FIG. 6 is a schematic diagram of a recombinant DNA fragment Ac96-Rep-CNE-Cap containing AAV Cap and Rep expression cassettes constructed in example 2 of the present invention.

FIG. 7 is a schematic diagram of a recombinant DNA fragment Ac96-Rep-NAE-Cap containing an AAV Cap and a Rep expression cassette constructed in example 2 of the present invention.

FIG. 8 is a green fluorescent plaque map generated after insect host cells were co-transfected with defective baculovirus vector Δ CNE-Bac and recombinant DNA fragment Ac96-CNE-GFP in example 3 of the present invention.

FIG. 9 is a graph showing the effect of green fluorescence expression of the recombinant baculovirus generated after infecting cells after transfecting insect host cells with defective baculovirus vector Δ CNE-Bac and recombinant DNA fragment Ac96-CNE-GFP in example 3 of the present invention.

FIG. 10 is a red fluorescent plaque map generated after co-transfection of insect host cells with defective baculovirus vector Δ NAE-Bac and recombinant DNA fragment Ac96-NAE-mcherry in example 3 of the present invention.

FIG. 11 is a graph showing the effect of red fluorescence expression of the recombinant baculovirus generated after infecting cells after transfecting insect host cells with defective baculovirus vector Δ NAE-Bac and recombinant DNA fragment Ac96-NAE-mcherry in example 3 of the present invention.

FIGS. 12A-F are schematic diagrams of recombinant DNA fragments with insertion sites at orf83, orf126 and orf152, respectively, constructed in example 4 of the present invention.

FIG. 13 is a green fluorescent plaque map generated by co-transfecting insect host cells with defective baculovirus vector Δ CNE-Bac and recombinant DNA fragments Ac83-CNE-GFP, Ac126-CNE-GFP and Ac152-CNE-GFP, respectively, in example 4 of the present invention. FIG. 14 is a diagram showing the effect of expressing green fluorescence after infecting cells with the generated recombinant baculovirus after transfecting insect host cells with defective baculovirus vector Δ CNE-Bac and recombinant DNA fragments Ac83-CNE-GFP, Ac126-CNE-GFP and Ac152-CNE-GFP, respectively, in example 4 of the present invention.

FIG. 15 is a red fluorescent plaque map generated after transfecting insect host cells with defective baculovirus vector Δ NAE-Bac and recombinant DNA fragments Ac83-NAE-mcherry, Ac126-NAE-mcherry and Ac152-NAE-mcherry, respectively, in example 4 of the present invention.

FIG. 16 is a graph showing the effect of red fluorescence expression after infection of insect host cells with the generated recombinant baculovirus after transfection of insect host cells with defective baculovirus vector Δ NAE-Bac and recombinant DNA fragments Ac83-NAE-mcherry, Ac126-NAE-mcherry and Ac152-NAE-mcherry, respectively, in example 4 of the present invention.

FIG. 17 is a Western Blot assay of expressed VP capsid and Rep proteins of example 5 of the present invention, which was performed by co-transfecting insect host cells with defective baculovirus vector Δ CNE-Bac and recombinant DNA fragment Ac 96-Rep-CNE-Cap.

FIG. 18 is a Western Blot analysis of expression of VP capsid and Rep proteins after co-transfection of insect host cells with defective baculovirus vector Δ NAE-Bac and recombinant DNA fragment Ac96-Rep-NAE-Cap in example 5 of the present invention.

FIG. 19 is a schematic diagram of a recombinant DNA fragment Ac83-ITR-NAE containing the AAV core expression element ITR-GOI constructed in example 8 of the present invention.

FIG. 20 is a silver stain image of SDS-PAGE of purified recombinant AAV virions after transfection of host cells with three recombinant baculoviruses of example 9 of the invention, showing three capsid proteins VP1/VP2/VP 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise. In the description of the present invention, "a plurality" means at least one, e.g., one, two, etc., unless specifically limited otherwise.

As used herein, an expression cassette refers to a nucleic acid construct comprising coding and regulatory sequences operably linked when introduced into a host cell, resulting in the transcription and/or translation of an RNA or polypeptide, respectively. An expression cassette is understood to include a promoter which allows transcription to begin, an open reading frame for the gene of interest, and a transcription terminator. Typically, the promoter sequence is placed upstream of the gene of interest, at a distance from the gene of interest that is compatible with expression control.

Cis-acting elements refer to specific DNA sequences in tandem with structural genes, which are binding sites for transcription factors that regulate the precise initiation of gene transcription and transcription efficiency by binding to transcription factors. Cis-acting elements include promoters, enhancers, regulatory sequences, inducible elements, and the like, which function to participate in the regulation of gene expression, do not encode any protein per se, and provide only one site of action.

AAV is a single-stranded DNA virus, the genome structure is simple, the total length is about 4.7kb, and the genome comprises a rep gene expression cassette, a cap gene expression cassette and AAV Inverted Terminal Repeats (ITRs) positioned at two ends of the genome. These are the three elements necessary for packaging AAV viruses. The Cap gene encodes a structural VP capsid protein comprising three overlapping open reading frames encoding three types of subunits, VP1, VP2, and VP3, respectively. The Rep gene encodes four overlapping multifunctional proteins Rep78, Rep68, Rep52 and Rep40, which are involved in replication and integration of AAV. The ITRs are 125 nucleotide palindromes at both ends of the genome, which form a self-complementary inverted-T hairpin structure, the cis-acting elements required for DNA replication initiation and packaging of recombinant AAV genomes into infectious viral particles. Since AAV is defective virus and cannot replicate independently in the absence of helper virus, AAV can only integrate into host cell chromosomes at a site and is latent. In the presence of helper virus, the increased rep gene expression can save AAV genome integrated in host cell chromosome, and obtain AAV DNA via mass replication, and the single-stranded rAAV genome is packaged into infectious virus particle under the action of VP capsid protein.

Conserved non-protein-coding elements (CNE) were found in the genomes of all sequenced baculovirus A (Alphabaculovirus), with a highly homologous sequence of 154-157 bp. It has been reported that an at-rich CNE sequence located in ac152 region of Autographa californica multiple nuclear polyhedrosis-virus (AcMNPV) genome is presumed to be a cis-acting element essential for the production of virus particles.

The NAE sequence was first discovered to be an essential element of nucleocapsid assembly in the genus baculovirus A, which plays an essential role in the nucleocapsid assembly process. The native NAE sequence was located proximally in and within the ac83 gene and its homologous genes in the A-type rhabdovirus (CN 106566829A). ac83 is core gene related to baculovirus nucleocapsid assembly, has a full length of 2544bp, encodes 847 amino acids, and has a predicted molecular weight of 96.2 kDa. Knock-out of ac83 did not affect viral genome replication, but completely blocked viral nucleocapsid assembly, and the presence of numerous hollow capsid precursors in the nucleus was observed under electron microscopy.

The invention provides a composition for producing a recombinant baculovirus in an insect cell, which comprises a packaging-defective baculovirus plasmid and a first rescue recombinant DNA, wherein the packaging-defective baculovirus plasmid is deleted of a CNE sequence and/or a NAE sequence in a baculovirus genome, the first rescue recombinant DNA comprises a first insertion sequence and a first homology arm positioned on two sides of the first insertion sequence, the first insertion sequence comprises a first functional fragment and a first complementary sequence, the first complementary sequence is at least one of the sequences deleted from the packaging-defective baculovirus plasmid, and the composition can be subjected to homologous recombination in the insect cell to produce the recombinant baculovirus.

It should be noted that the packaging-defective baculovirus plasmid according to the present invention cannot normally package a recombinant baculovirus because of deletion of CNE sequence and/or NAE sequence in the baculovirus genome. The CNE sequence can be a CNE sequence that is identical to a wild-type AcMNPV CNE sequence, can also be a CNE sequence from other baculoviruses, or an artificial CNE sequence that shares at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or higher sequence identity with a wild-type AcMNPV CNE sequence. Similarly, the NAE sequence can be an NAE sequence that is identical to a wild-type AcMNPV NAE sequence, can be an NAE sequence from other baculoviruses, or can be an artificial NAE sequence that shares at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or greater sequence identity with a wild-type AcMNPV NAE sequence.

Furthermore, when the packaging-defective baculovirus plasmid lacks a CNE sequence in the baculovirus genome, the corresponding, first complementing sequence is a CNE sequence; when the packaging defective baculovirus plasmid lacks an NAE sequence in the baculovirus genome, the first complementing sequence is an NAE sequence; when the packaging-defective baculovirus plasmid simultaneously deletes the CNE sequence and the NAE sequence in the baculovirus genome, the first complementing sequence accordingly contains both the CNE sequence and the NAE sequence. Thus, the recombinant bacmid after homologous recombination in insect cells contains normal CNE and NAE sequences, and further can package recombinant baculovirus; and when homologous recombination does not occur, the CNE or NAE sequence is deleted on the bacmid, so that the recombinant baculovirus cannot be normally packaged. In another example, the second rescue recombinant DNA and the first rescue recombinant DNA can also be used to make up for the deleted sequence, i.e., when the packaging-defective baculovirus plasmid simultaneously deletes both the CNE sequence and the NAE sequence in the baculovirus genome, the second rescue recombinant DNA comprises a second insertion sequence comprising a second anaplerotic sequence, the first anaplerotic sequence being either the CNE sequence or the NAE sequence, and a second homology arm flanking the second insertion sequence, the second anaplerotic sequence being a different one of the CNE sequence and the NAE sequence than the first anaplerotic sequence. It is noted that the first and second homology arms herein are homologous sequences corresponding to different loci on the baculovirus genome, such that the first and second insertion sequences are capable of being inserted into different loci on the baculovirus genome.

It should be noted that the rescue recombinant DNA of the present invention may be a linear DNA fragment or a baculovirus transfer vector, and is not limited herein. In addition, the rescue recombinant DNA provided by the invention has no limitation of an insertion site, and when the rescue recombinant DNA and the packaging-defective baculovirus plasmid are subjected to homologous recombination, the insertion sequence on the rescue recombinant DNA can be inserted into any gene locus of the baculovirus genome, such as but not limited to Ac18, Ac83, Ac96, Ac126, Ac127, Ac130 and Ac 152.

The first functional fragment in the invention can be a cap gene expression cassette of AAV and a rep gene expression cassette of AAV, and can also be an AAV ITR core expression element (namely ITR-GOI) with exogenous genes, and is used for preparing recombinant adeno-associated virus. The foreign Gene may be at least one nucleotide sequence encoding a Gene of Interest (GOI) product, which may be a therapeutic Gene product, in particular a polypeptide, an RNA molecule (siRNA) or other Gene product, such as but not limited to lipoprotein esterase, apolipoprotein, cytokine, interleukin or interferon; it may also be a reporter protein to assess vector transformation and expression, such as but not limited to fluorescent proteins (green fluorescent protein, GFP, red fluorescent protein, RFP), chloramphenicol acetyltransferase, β -galactosidase, β -glucuronidase, renilla luciferase, firefly luciferase or alkaline phosphatase. Furthermore, the first functional fragment may also be a reporter gene, such as but not limited to a gene sequence expressing a fluorescent protein (green fluorescent protein GFP, red fluorescent protein RFP), chloramphenicol acetyltransferase, β -galactosidase, β -glucuronidase, renilla luciferase, firefly luciferase or alkaline phosphatase, for the purpose of evaluating the validation of the occurrence of homologous recombination to produce a recombinant baculovirus. The first functional fragment may also be any nucleotide sequence encoding a therapeutic gene product, such as but not limited to, a nucleotide sequence encoding a pharmaceutical polypeptide (e.g., interleukin, etc.) or a viral recombinant subunit protein.

In the present embodiment, the specific positions and orientations of the cap gene expression cassette and the rep gene expression cassette are not limited, and the arrangement of the cap gene expression cassette, the rep gene expression cassette, and the first complementing sequence may be six: 1. a cap gene expression box, a rep gene expression box and a first complementing sequence are sequentially arranged from the 5 'end to the 3' end; 2. sequentially arranging a rep gene expression box, a cap gene expression box and a first complementing sequence from a 5 'end to a 3' end; 3. sequentially arranging a cap gene expression box, a first complementary sequence and a rep gene expression box from a 5 'end to a 3' end; 4. sequentially arranging a rep gene expression box, a first complementary sequence and a cap gene expression box from a 5 'end to a 3' end; 5. sequentially arranging a first complementing sequence, a cap gene expression cassette and a rep gene expression cassette from a 5 'end to a 3' end; 6. the first complementing sequence, the rep gene expression cassette and the cap gene expression cassette are arranged in sequence from the 5 'end to the 3' end. The cap gene expression cassette and the rep gene expression cassette may be in the same orientation or in opposite orientations. The sequence of the cap gene expression cassette in the recombinant expression cassette may be from 5 'to 3' or from 3 'to 5'. Similarly, the rep gene expression cassette may be in the order of the recombinant expression cassette from 5 'to 3' or from 3 'to 5'.

As a preferred example, the first complementing sequence is located between the cap gene expression cassette and the rep gene expression cassette, specifically, the first insertion sequence comprises the cap gene expression cassette, the CNE sequence and the rep gene expression cassette in the order from 5 'to 3', or the first insertion sequence comprises the cap gene expression cassette, the NAE sequence and the rep gene expression cassette in the order from 5 'to 3', or the first insertion sequence comprises the rep gene expression cassette, the CNE sequence and the cap gene expression cassette in the order from 5 'to 3', or the rep gene expression cassette, the NAE sequence and the cap gene expression cassette in the order from 5 'to 3'. Further preferably, both ends of the first complementing sequence are close to the initiating end of the cap gene expression cassette and the initiating end of the rep gene expression cassette, respectively, that is, the cap gene expression cassette and the rep gene expression cassette are in opposite directions, and the initiating ends of the cap gene expression cassette and the rep gene expression cassette are oppositely arranged and face the first complementing sequence.

The invention provides an insect cell comprising any one of the compositions.

The present invention provides a method for in vitro growth or production of a recombinant baculovirus, comprising co-transfecting an insect cell with any one of the compositions described above and culturing the insect cell. The recombinant baculovirus can then be recovered.

The present invention also provides a method for in vitro growth or production of recombinant adeno-associated virus, comprising co-transfecting insect cells with the composition and culturing the insect cells to produce the recombinant adeno-associated virus. It is emphasized that the compositions required for the production of recombinant adeno-associated viruses need to contain the AAV cap gene, rep gene and ITR core expression elements necessary for rAAV production, and thus, if the first rescue recombinant DNA in the composition comprises an AAV cap gene expression cassette and an AAV rep gene expression cassette, it is also necessary to insert an AAV ITR core expression element with a foreign gene into the packaging-defective baculovirus plasmid, which can be obtained by baculovirus transfer vector-mediated Tn7 transposition. If the first rescue recombinant DNA in the composition contains an AAV ITR core expression element with a foreign gene, it is also necessary to insert a cap gene expression cassette of AAV and a rep gene expression cassette of AAV, which can be obtained by baculovirus transfer vector-mediated transposition of Tn7, into the packaging-defective baculovirus plasmid. In addition, the aim of obtaining rAAV can be achieved by two rescue recombinant DNAs, the package-defective baculovirus plasmid simultaneously deletes the CNE sequence and the NAE sequence in the baculovirus genome, for example, the first rescue recombinant DNA comprises a cap gene expression cassette of AAV, a rep gene expression cassette of AAV and the CNE sequence, and the second rescue recombinant DNA comprises an AAV ITR core expression element with a foreign gene and the NAE sequence.

The above technical solution is described in detail below with reference to specific examples.

Example 1 construction of a CNE sequence-deleted bacmid-DeltaCNE-Bac, a NAE sequence-deleted bacmid-DeltaNAE-Bac, and a CNE and NAE sequence-deleted bacmid-DeltaCNE-DeltaNAE-Bac

Red recombination is a highly efficient recombination method at the bacterial level and can be used to rapidly engineer recombinant baculovirus genomes in Escherichia coli (DH10 Bac). The Red recombination is to use lambda phage Red recombinase (composed of 3 proteins of Exo, Beta and Gam) to carry out homologous recombination on a linear DNA fragment which is introduced into a cell and carries a homology arm and a specific target sequence of a genome so as to realize the replacement of a target gene (Doublet et al, 2008, J Microbiol Methods, 75 (2): 359-361).

Firstly, constructing a first homologous recombination expression frame (SEQ ID No.1) of a targeted CNE sequence, wherein the expression frame sequentially comprises a CNE upstream homologous sequence, a chloramphenicol (Chol) resistance gene expression frame and a CNE downstream homologous sequence from 5 'to 3' as shown in figure 1; then, the expression frame and the CNE sequence on the bacmid are replaced by utilizing a Red recombination technology, so that the bacmid delta CNE-Bac with the CNE sequence deleted is obtained.

Similarly, a second homologous recombination expression frame (SEQ ID No.2) of the targeted NAE sequence is firstly constructed, and as shown in figure 2, the expression frame sequentially comprises an NAE upstream homologous sequence, a chloramphenicol (Chol) resistance gene expression frame and an NAE downstream homologous sequence from 5 'to 3'; then, the expression frame is replaced with the NAE sequence on the bacmid by utilizing the Red recombination technology, thereby obtaining the bacmid delta NAE-Bac with the deleted NAE sequence.

Similarly, first, a third homologous recombination expression cassette (SEQ ID No.3) targeting the NAE sequence was constructed, which comprises, in order from 5 'to 3', an NAE upstream homologous sequence, a Gentamicin (GM) resistance gene expression cassette, and an NAE downstream homologous sequence, as shown in FIG. 3; then, the expression frame is replaced with the NAE sequence on the bacmid delta CNE-Bac by utilizing the Red recombination technology, thereby obtaining the bacmid delta CNE-delta NAE-Bac with the CNE and NAE sequences deleted simultaneously.

EXAMPLE 2 construction of recombinant DNA transfer vectors containing CNE or NAE sequences

In the embodiments of the invention, Green Fluorescent Protein (GFP) or red fluorescent protein (mcherry) is used as an exogenous gene to be inserted into an AcMNPV genome. Constructing a first recombinant DNA fragment with an insertion site for a foreign gene at the AcMNPVorf96 locus: referring to FIG. 4, the recombinant DNA fragment comprises an upstream homologous sequence of orf96, a CNE sequence (SEQ ID No.4), a GFP expression cassette and a downstream homologous sequence of orf96 in sequence from 5 'to 3', and the sequences are connected by artificial direct synthesis or overlap extension PCR amplification to obtain a construct Ac96-CNE-GFP respectively. Similarly, construct Ac96-NAE-mcherry (FIG. 5) containing the NAE sequence (SEQ ID No.5) was constructed. The nucleotide sequences of the construct Ac96-CNE-GFP and the construct Ac96-NAE-mcherry are shown in SEQ ID No.6 and SEQ ID No.7, respectively.

Construction of a second recombinant DNA fragment with insertion sites for AAV rep and cap gene expression cassettes at the AcMNPVorf96 locus: referring to FIG. 6, the recombinant DNA fragment sequentially comprises an orf96 upstream homologous sequence, a Rep gene expression cassette (SEQ ID No.8), a CNE sequence, a Cap gene expression cassette (SEQ ID No.9) and an orf96 downstream homologous sequence from 5 'to 3', and the sequences are artificially synthesized directly or amplified and connected by overlap extension PCR to obtain a construct Ac 96-Rep-CNE-Cap. Similarly, construct Ac96-Rep-NAE-Cap containing NAE sequences was constructed (FIG. 7). The nucleotide sequences of the construct Ac96-Rep-CNE-Cap and the construct Ac96-Rep-NAE-Cap are shown in SEQ ID No.10 and SEQ ID No.11, respectively.

Example 3 verification of production of recombinant baculovirus by homologous recombination after Co-transfection of insect host cells with packaging deficient baculovirus vectors and recombinant DNA fragments

In this example, Sf9 insect host cells were co-transfected with the packaging-defective baculovirus vectors Δ CNE-Bac and Δ NAE-Bac constructed in example 1 and the corresponding recombinant DNA fragments Ac96-CNE-GFP and Ac96-NAE-mcherry constructed in example 2, respectively, and after 96h of co-transfection, whether the host cells can produce green fluorescent plaques or red fluorescent plaques was observed by using a fluorescence microscope to determine the production of recombinant baculovirus. To confirm that the co-transfection indeed produced recombinant baculovirus, the culture supernatant of Sf9 cells was collected 120h after co-transfection, transferred to host cells in suspension culture, and observed for green fluorescence or red fluorescence emission with a fluorescence microscope 72h after infection.

The observation results are shown in fig. 8 to 11: the baculovirus vector delta CNE-Bac and the recombinant DNA fragment Ac96-CNE-GFP transfect Sf9 cells for 96h to generate green fluorescent plaques (figure 8), after the cotransfection is carried out for 120h, culture supernatant of Sf9 cells is collected and transferred to host cells cultured in a suspension manner, and a large amount of green fluorescence is generated after infection is carried out for 72h (figure 9); the baculovirus vector delta NAE-Bac and the recombinant DNA fragment Ac96-NAE-mcherry transfect Sf9 cells for 96h to generate red fluorescent plaques (figure 10), and after 120h of cotransfection, culture supernatant of Sf9 cells is collected and transferred to host cells cultured in suspension, and a large amount of red fluorescence is generated after 72h of infection (figure 11). This example shows that package-defective baculovirus vector Δ CNE-Bac or Δ NAE-Bac and recombinant DNA fragment co-transfect insect host cell and then can generate homologous recombination to generate recombinant baculovirus, and perform protein expression of exogenous gene.

Example 4 foreign genes can be inserted into any locus of AcMNPV genome without restriction of insertion site, and can be inserted into target site according to requirement

First, recombinant DNA fragments having insertion sites at orf83, orf126 and orf152, respectively, were constructed as shown in FIGS. 12A-F with reference to example 2: wherein the constructs Ac83-CNE-GFP (FIG. 12A), Ac126-CNE-GFP (FIG. 12B) and Ac152-CNE-GFP (FIG. 12C) are recombinant DNA fragments with insertion sites with CNE complement sequences at orf83, orf126 and orf152, respectively, and the constructs Ac83-NAE-mcherry (FIG. 12D), Ac126-NAE-mcherry (FIG. 12E) and Ac152-NAE-mcherry (FIG. 12F) are recombinant DNA fragments with insertion sites with NAE complement sequences at orf83, orf126 and orf152, respectively;

in this example, the packaging-defective baculovirus vectors Δ CNE-Bac and Δ NAE-Bac of example 1 were co-transfected with the recombinant DNA fragment of this example into Sf9 insect host cells, respectively, and after co-transfection for 96 hours, the production of recombinant baculovirus was determined by observing whether the host cells could produce green fluorescent plaques or red fluorescent plaques using a fluorescence microscope. To confirm that the co-transfection did indeed produce recombinant baculovirus, after 120h of co-transfection, culture supernatant of Sf9 cells was collected, transferred to host cells in suspension culture, and observed for green fluorescence or red fluorescence emission with a fluorescence microscope 72h after infection.

The observation results are shown in fig. 13 to 16: the baculovirus vector delta CNE-Bac cotransfects Sf9 cells with recombinant DNA fragments Ac83-CNE-GFP, Ac126-CNE-GFP and Ac152-CNE-GFP respectively for 96h to generate green fluorescent plaques (figure 13), collects culture supernatant of Sf9 cells after cotransfection for 120h, transfers the culture supernatant to host cells cultured in suspension, and generates a large amount of green fluorescence after infection for 72h (figure 14); the baculovirus vector delta NAE-Bac co-transfects Sf9 cells with recombinant DNA fragments Ac83-NAE-mcherry, Ac126-NAE-mcherry and Ac152-NAE-mcherry respectively for 96h to generate red fluorescent plaques (figure 15), collects culture supernatant of Sf9 cells after 120h of co-transfection, transfers the culture supernatant to host cells cultured in suspension, and generates a large amount of red fluorescence after 72h of infection (figure 16). This example shows that a foreign gene can be inserted into any locus of the AcMNPV genome without restriction of the insertion site, and can be inserted into a target site as needed to express the foreign gene.

EXAMPLE 5 obtaining recombinant baculovirus containing Cap and Rep, elements essential for AAV packaging, and detecting expression of Cap and Rep

Sf9 insect cells were co-transfected with the packaging defective baculovirus vector Δ CNE-Bac in example 1 and the recombinant DNA fragment Ac96-Rep-CNE-Cap in example 2, and Sf9 insect cells were co-transfected with the packaging defective baculovirus vector Δ NAE-Bac in example 1 and the recombinant DNA fragment Ac96-Rep-NAE-Cap in example 2, respectively, to prepare recombinant baculovirus BEV. After transfection, Sf9 insect cells successfully produced BEV, and further infection of BEV with mass replication and proliferation resulted in obvious cytopathic effect (CPE) of Sf9 cells. Collecting the culture supernatant of Sf9 cells with CPE, wherein the culture supernatant contains a large amount of BEV, namely BEV 0 (BEV-P0), and collecting Sf9 cells containing a large amount of rAAV. The prepared BEV-P0 was infected with Sf9 cells cultured in suspension at multiplicity of infection (MOI ═ 1), cell viability was reduced to 50% or less after 72 hours of infection, 1000g of the cell culture broth was centrifuged for 5min, and culture supernatant and cell pellet were collected, respectively, and the supernatant was labeled as BEV generation 1 (BEV-P1). After the scale-up culture was continued, the suspension cultured Sf9 cells were infected with BEV-P1 at multiplicity of infection (MOI ═ 1), and 72 hours after infection, the cell activity was reduced to 50% or less, and the cell culture broth was centrifuged at 1000g for 5min to collect cell pellets, and Western Blot was performed to detect the expression of VP proteins (VP1, VP2, and VP3) and Rep proteins (Rep78 and Rep 52).

The results are shown in fig. 17 and 18: after the baculovirus vector Delta CNE-Bac and the recombinant DNA fragment Ac96-Rep-CNE-Cap transfect Sf9 insect cells together, recombinant baculovirus is generated, and VP protein and Rep protein are successfully expressed (figure 17); the recombinant baculovirus was generated by co-transfecting Sf9 insect cells with the baculovirus vector Δ NAE-Bac and the recombinant DNA fragment Ac96-Rep-NAE-Cap, and successfully expressed the VP protein and Rep protein (FIG. 18).

EXAMPLE 6 construction of recombinant baculovirus vectors containing AAV core expression element (ITR-GOI)

This example method for constructing recombinant AAV bacmid for production of AAV virus in insect cells reference example 1 of the applicant's previous application patent CN112553257A, comprising the following steps:

(1) construction of a recombinant transfer vector comprising an ITR core element (ITR-GOI). The nucleotide sequence of ITR-GOI is shown as SEQ ID No. 12. In the embodiment, the GOI in the ITR core element adopts a red fluorescent protein mcherry gene expression cassette, namely, the micherry expression is controlled by a miniEf1a promoter, so that the activity of rAAV can be conveniently detected, and the ITR and the red fluorescent protein expression cassette are constructed on a transfer vector pFastDual.

(2) And (2) transforming competent cells containing delta CNE-Bac or delta NAE-Bac bacmid by using the recombinant transfer vector constructed in the step (1), inserting ITR-GOI into Tn7 site of the delta CNE-Bac or the delta NAE-Bac bacmid by using Tn7 recombination, and finally obtaining recombinant baculovirus plasmids containing ITR core elements necessary for producing rAAV, wherein the numbers of the recombinant baculovirus plasmids are delta CNE-Bac-Tn7-ITR and delta NAE-Bac-Tn7-ITR respectively.

EXAMPLE 7 preparation of AAV recombinant baculoviruses containing the AAV packaging essential elements Cap, Rep and ITR-GOI Using either a DeltaCNE-Bac-Tn 7-ITR or a DeltaNAE-Bac-Tn 7-ITR defective bacmid

Respectively co-transfecting the recombinant bacmid-Bac-Tn 7-ITR prepared in the example 6 and the recombinant DNA fragment Ac97-Rep-CNE-Cap prepared in the example 2, and the recombinant bacmid-Bac-Tn 7-ITR prepared in the example 6 and the recombinant DNA fragment Ac97-Rep-NAE-Cap prepared in the example 2 to obtain AAV recombinant baculoviruses, wherein the numbers of the recombinant bacmid-Bac-AAV and the recombinant DNA fragment Ac-Bac-AAV are respectively as follows:

extracting the recombinant bacmid and the transfer vector DNA to co-transfect Sf9 insect cells and preparing the recombinant baculovirus BEV and rAAV. After co-transfection Sf9 insect cells successfully produced BEV, further infection of the large number of replicating proliferating BEV resulted in a clear cytopathic effect (CPE) in Sf9 cells. Culture supernatants of Sf9 cells with CPE were collected, containing large amounts of BEV, i.e., BEV generation 0 (BEV-P0), while Sf9 cells containing large amounts of rAAV were collected. The prepared BEV-P0 was infected with Sf9 cells in suspension culture at multiplicity of infection (MOI ═ 1), the cell activity was reduced to 50% or less after 72 hours of infection, 1000g of the cell culture broth was centrifuged for 5min, and culture supernatant and cell pellet were collected, respectively, and the supernatant was labeled as BEV (BEV-P1) of the 1 st generation and the cells were labeled as rAAV packaged in BEV-P0.

EXAMPLE 8 preparation of AAV recombinant baculoviruses containing the AAV packaging essential elements Cap, Rep and ITR-GOI Using a DeltaCNE-DeltaNAE-Bac defective bacmid

First, a recombinant DNA fragment was constructed with the insertion site for AAV ITR core expression element (ITR-GOI) at the AcMNPV orf83 locus: referring to FIG. 19, the recombinant DNA fragment sequentially comprises an orf83 upstream homologous sequence, an ITR-GOI sequence, an NAE sequence and an orf83 downstream homologous sequence from 5 'to 3', and the sequences are connected through artificial direct synthesis or overlap extension PCR amplification to obtain a construct Ac 83-ITR-NAE.

Then, the defective bacmid-DeltaCNE-DeltaNAE-Bac prepared in example 1 was co-transfected with the recombinant DNA fragment Ac97-Rep-CNE-Cap prepared in example 2 and the recombinant DNA fragment Ac83-ITR-NAE prepared in this example to obtain AAV recombinant baculovirus, numbered DeltaCNE-DeltaNAE-Bac-AAV, according to the specific procedure of example 7.

Example 9 purification of recombinant AAV virions and detection of packaging efficiency thereof

Recombinant baculoviruses ac-Bac-AAV, ac-Bac-AAV and ac-CNE-ac-Bac-AAV in examples 7 and 8 were further grown up in scale-up according to the procedure of example 5 until suspension cultured Sf9 cells were infected with a seed virus of BEV-P2 at multiplicity of infection (MOI ═ 1) for packaging of rAAV in a package volume of 300mL to 400 mL. Monitoring cell activity 3 days after infection, wherein the activity is lower than 50%, respectively centrifuging to obtain cell sediment and supernatant, respectively purifying the obtained cell sediment and supernatant, repeatedly freezing and thawing the cells for 3 times for lysis, centrifuging at 5000rpm for 10min, collecting supernatant, adding nuclease (Benzonase) into the supernatant, treating in 37 deg.C water bath for 60min, and centrifuging at 5000rpm for 10 min. The collected cell lysate and the collected supernatant were PEG precipitated, resuspended and purified by iodixanol density gradient centrifugation (see Aslanidii et al, 2009, Proc. Natl. Acad. Sci. USA,206: 5059-5064). The final purified finished virus was resuspended in 80. mu.L-190. mu.L PBS, 10. mu.L purified finished virus was run on SDS-PAGE gel and silver stained.

The SDS-PAGE gel is shown in FIG. 20: wherein, lane 1 is the silver staining test chart of SDS-PAGE of purified recombinant AAV virions after recombinant baculovirus delta CNE-Bac-AAV infects host cells, showing three capsid proteins VP1/VP2/VP 3; lane 2 is a silver stain assay of purified recombinant AAV virions on SDS-PAGE after infection of host cells with recombinant baculovirus Δ NAE-Bac-AAV, showing three capsid proteins VP1/VP2/VP 3; lane 3 is a silver stain assay of purified recombinant AAV virions on SDS-PAGE after infection of host cells with recombinant baculovirus Δ CNE- Δ NAE-Bac-AAV, showing three capsid proteins VP1/VP2/VP 3.

This example also employed Q-PCR to determine the packaging rate of the harvested rAAV virus using a pair of primers targeting the ITR sequence (Q-ITR-F: GGAACCCCTAGTGATGGAGTT and Q-ITR-R: CGGCCTCAGTGAGCGA). The results are shown in Table 1.

TABLE 1 detection result table of rAAV virions produced by recombinant baculovirus

Recombinant baculovirus numbering	Cell packing Rate (VG/cell)
		△CNE-Bac-AAV	4.83E+05
△NAE-Bac-AAV	5.26E+05
		△CNE-△NAE-Bac-AAV	4.85E+05

This example shows that the defective baculovirus vector and recombinant DNA fragment provided by the present invention can be directly recombined in insect host cells to prepare AAV recombinant baculovirus containing the AAV packaging essential elements Cap, Rep and ITR-GOI, and successfully produce AAV virions.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

SEQUENCE LISTING

<110> Rui Zheng medical science and technology (Wuhan) Co., Ltd

<120> a composition, method and use for generating recombinant baculovirus in insect cells

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 1590

<212> DNA

<213> Artificial Sequence

<220>

<223> Expression cassette homologous recombinant

<400> 1

tgctcctgac gccgctcctg acggcgatgg ctgcgactgc ttgaagacgg ctggctgcga 60

ctgcttgaag acggctgggc ttcgggagat gttgtaaagt tgatgcggcg acggctgaga 120

gacagcctgt ggcggcggct gctgctggga gtggcggcgt tgatttggcg actcatggct 180

gggctggtag gatactgttc actaggctgt gaggcttgaa ctgtgcttac gagtagaacg 240

gcagctgtat ttatactgtt tatcagtact gcacgactga taagacaata gtggtggggg 300

aacttgccag gcaaaaatga gaagttccta tactttctag agaataggaa cttcatttaa 360

atggcgcgcc ttacgccccg ccctgccact catcgcagta ctgttgtatt cattaagcat 420

ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc 480

accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc 540

atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa 600

aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca 660

tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 720

gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 780

accagctcac cgtctttcat tgccatacgt aattccggat gagcattcat caggcgggca 840

agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 900

gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc 960

tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt 1020

ttctccattt tagcttcctt agctcctgaa aatctcgaca actcaaaaaa tacgcccggt 1080

agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat 1140

tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat 1200

tctgcgaagt gatcttccgt cacaggtagg cgcgccgaag ttcctatact ttctagagaa 1260

taggaacttc tcattttaaa tttatcatat cacaggctgc agtttctgtt atctgtcccc 1320

cactcaggcg tgcagctata aaagcaggca ctcaccaact cgtaagcaca gttcgttgtg 1380

aagtgaacac ggagagcctg ccaataagca aaatgccaag ggacaccaac aatcgccacc 1440

ggtctacgcc atatgaacgt cctacgcttg aagatctccg cagacagttg caagacaatt 1500

tggacagcat aaaccgccga gacagaatgc aagaagaaca agaagaaaac ctgcgctatc 1560

aagtgcgtag aaggcagcgt caaaaccagc 1590

<210> 2

<211> 1277

<212> DNA

<213> Artificial Sequence

<220>

<223> Expression cassette homologous recombinant

<400> 2

cacccgacgt aataggctac gtgtcggata ttatgcaaaa cacttatatt gtaacgtggt 60

tcaacaccgt cgacctttcc acctatcacg aaagcgtgca tgatgaccgg attgaaattt 120

ttgatttctt aaatcaaaaa tttcaacctg ttgatcgaat cgtacacgat cgcgttagag 180

caaatgatga aaatcccaac gaagttccta tactttctag agaataggaa cttcatttaa 240

atggcgcgcc ttacgccccg ccctgccact catcgcagta ctgttgtatt cattaagcat 300

ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc 360

accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc 420

atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa 480

aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca 540

tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 600

gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 660

accagctcac cgtctttcat tgccatacgt aattccggat gagcattcat caggcgggca 720

agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 780

gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc 840

tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt 900

ttctccattt tagcttcctt agctcctgaa aatctcgaca actcaaaaaa tacgcccggt 960

agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat 1020

tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat 1080

tctgcgaagt gatcttccgt cacaggtagg cgcgccgaag ttcctatact ttctagagaa 1140

taggaacttc gcacgtcaaa aacggccaat acatggcgtg tcccgaagaa ttgtacgata 1200

acaacgaatt taaatgtaac atagaatcgg ataaattata ctatttggat aatttacaag 1260

aagattccat tgtataa 1277

<210> 3

<211> 1528

<212> DNA

<213> Artificial Sequence

<220>

<223> Expression cassette homologous recombinant

<400> 3

atgatgtctg gcgtaatgtt gctcatgctt gccatttttc ttataatagc gtttacttta 60

atgtatttgg caatttattt tgaatttgac gaaacgactt tcaccaagcg gctccaagtg 120

atgactgaat atgtgaagcg caccaacgca gacgaaccca cacccgacgt aataggctac 180

gtgtcggata ttatgcaaaa cacttatatt gtaacgtggt tcaacaccgt cgacctttcc 240

acctatcacg aaagcgtgca tgatgaccgg attgaaattt ttgatttctt aaatcaaaaa 300

tttcaacctg ttgatcgaat cgtacacgat cgcgttagag caaatgatga aaatcccaac 360

ataacttcgt ataatttata ctatacgaag ttatatttaa atttaggtgg cggtacttgg 420

gtcgatatca aagtgcatca cttcttcccg tatgcccaac tttgtataga gagccactgc 480

gggatcgtca ccgtaatctg cttgcacgta gatcacataa gcaccaagcg cgttggcctc 540

atgcttgagg agattgatga gcgcggtggc aatgccctgc ctccggtgct cgccggagac 600

tgcgagatca tagatataga tctcactacg cggctgctca aacctgggca gaacgtaagc 660

cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca agcgcgatga atgtcttact 720

acggagcaag ttcccgaggt aatcggagtc cggctgatgt tgggagtagg tggctacgtc 780

tccgaactca cgaccgaaaa gatcaagagc agcccgcatg gatttgactt ggtcagggcc 840

gagcctacat gtgcgaatga tgcccatact tgagccacct aactttgttt tagggcgact 900

gccctgctgc gtaacatcgt tgctgctgcg taacatttta gcttccttag ctcctgaaaa 960

tctcgacaac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga 1020

acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg gcttcccggt 1080

atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca caggtaggcg 1140

cgccataact tcgtataatt tatactatac gaagttatgc acgtcaaaaa cggccaatac 1200

atggcgtgtc ccgaagaatt gtacgataac aacgaattta aatgtaacat agaatcggat 1260

aaattatact atttggataa tttacaagaa gattccattg tataaacatt ttatgtcgaa 1320

aacaaatgac atcattccgg atcatgattt acgcgtagaa ttctacttgt aaagcaagtt 1380

aaaataagcc gtgtgcaaaa atgacatcag acaaatgaca tcatctacct atcatgatca 1440

tgttaataat catgttttaa aatgacatca gcttatgact aataattgat cgtgcgttac 1500

aagtagaatt ctactcgtaa agcgagtt 1528

<210> 4

<211> 156

<212> DNA

<213> Artificial Sequence

<220>

<223> CNE

<400> 4

acttttttgt aatgcaaaaa agttgatagt gtagtagtat attgggagcg tatcgtacag 60

tgtagactat tctaataaaa tagtctacga tttgtagaga ttgtactgta tatggagtgt 120

caggcaaaag tgaacttttt tgcattgcaa aaaaat 156

<210> 5

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<223> NAE

<400> 5

cattttcagc gacgtatatt gacaaatata ctacagtcgg acgtttgtgc cgacctatat 60

actacacttt accaaaaata tactacacta aactctaaat atactacaac tccacttcaa 120

tataaccaca ctctcgtaaa acggcccaaa aatatcgaaa tatatggggc aaatacacgt 180

ttaaaaaacg ctacgattcc 200

<210> 6

<211> 2254

<212> DNA

<213> Artificial Sequence

<220>

<223> Ac96-CNE-GFP

<400> 6

tcaaaaaaaa ttgtaaaatg ttgtcaatca tgttggctat cgtgtttgta cttttcgtgt 60

taatttattt aataatttcg atcaaaaatc accatccatt cttacataga atagaaacgc 120

taatacaaga tttcaacaac acattgttgt ttggcgcgta tgtacagatt tacgatttaa 180

gcacgcccgc ccgcaccgaa cgattgttta ttattgcgcc cgaaaatgtg gtgttgtata 240

attttaacaa aacgctctat tattacttgg actcggcgaa cgtgttttgt cccaacgagt 300

ttagcgtgac cacgttcacg caatccacta ttaaaacgat caacgagacg ggaatatatg 360

ccaccgcatg cacgccggtc agcagcttga cgctaattga acattttgca acattaaaaa 420

ataacgtgcc cgatcacacg ctcgttctcg cctaggctca agcagtgatc agatccagac 480

atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 540

tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 600

caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag 660

gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagta 720

cttctcgaca agcttggatc cgcgcccgat ggtgggacgg tatgaataat ccggaatatt 780

tataggtttt tttattacaa aactgttacg aaaacagtaa aatacttatt tatttgcgag 840

atggttatca ttttaattat ctccatgatc tattaatatt ccggaatttt tttgcaatgc 900

aaaaaagttc acttttgcct gacactccat atacagtaca atctctacaa atcgtagact 960

attttattag aatagtctac actgtacgat acgctcccaa tatactacta cactatcaac 1020

ttttttgcat tacaaaaaag tgtatacgga cctttaattc aacccaacac aatatattat 1080

agttaaataa gaattattat caaatcattt gtatattaat taaaatacta tactgtaaat 1140

tacattttat ttacaatcac tcgacgaaga cttgatcacc cgggatggtg agcaagggcg 1200

aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1260

acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 1320

agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 1380

cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 1440

agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 1500

actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 1560

tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 1620

acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 1680

tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 1740

acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 1800

ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 1860

ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaggtacc gggagatggg 1920

ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa 1980

taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcatgtggt cgaccaacag 2040

attcagtttt caatactcga cattatcaat tatttgattt acaatggcta cgtggatttg 2100

ttggccgaat aacgcgtata tagacgcttg tacgttcatc gtagtaatca ttttaataca 2160

tttgattgaa ctaaacatac atctgcaatg ggtgaaagag tcactaaatt ttgcaatgga 2220

aaacggcgat aaagaagaca gcgacaatga atag 2254

<210> 7

<211> 2289

<212> DNA

<213> Artificial Sequence

<220>

<223> Ac96-NAE-mcherry

<400> 7

tcaaaaaaaa ttgtaaaatg ttgtcaatca tgttggctat cgtgtttgta cttttcgtgt 60

taatttattt aataatttcg atcaaaaatc accatccatt cttacataga atagaaacgc 120

taatacaaga tttcaacaac acattgttgt ttggcgcgta tgtacagatt tacgatttaa 180

gcacgcccgc ccgcaccgaa cgattgttta ttattgcgcc cgaaaatgtg gtgttgtata 240

attttaacaa aacgctctat tattacttgg actcggcgaa cgtgttttgt cccaacgagt 300

ttagcgtgac cacgttcacg caatccacta ttaaaacgat caacgagacg ggaatatatg 360

ccaccgcatg cacgccggtc agcagcttga cgctaattga acattttgca acattaaaaa 420

ataacgtgcc cgatcacacg ctcgttctcg cctaggctca agcagtgatc agatccagac 480

atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 540

tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 600

caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag 660

gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagta 720

cttctcgaca agcttggatc cgcgcccgat ggtgggacgg tatgaataat ccggaatatt 780

tataggtttt tttattacaa aactgttacg aaaacagtaa aatacttatt tatttgcgag 840

atggttatca ttttaattat ctccatgatc tattaatatt ccggacattt tcagcgacgt 900

atattgacaa atatactaca gtcggacgtt tgtgccgacc tatatactac actttaccaa 960

aaatatacta cactaaactc taaatatact acaactccac ttcaatataa ccacactctc 1020

gtaaaacggc ccaaaaatat cgaaatatat ggggcaaata cacgtttaaa aaacgctacg 1080

attccgtata cggaccttta attcaaccca acacaatata ttatagttaa ataagaatta 1140

ttatcaaatc atttgtatat taattaaaat actatactgt aaattacatt ttatttacaa 1200

tcactcgacg aagacttgat cacccgggat ggtgagcaag ggcgaggagg ataacatggc 1260

catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga 1320

gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct 1380

gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat 1440

gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc 1500

cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac 1560

cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg 1620

caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc 1680

ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa 1740

gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc 1800

cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga 1860

ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat 1920

ggacgagctg tacaagtaag gtaccgggag atgggggagg ctaactgaaa cacggaagga 1980

gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg 2040

gtgttgggtc gtttgttcat gtggtcgacc aacagattca gttttcaata ctcgacatta 2100

tcaattattt gatttacaat ggctacgtgg atttgttggc cgaataacgc gtatatagac 2160

gcttgtacgt tcatcgtagt aatcatttta atacatttga ttgaactaaa catacatctg 2220

caatgggtga aagagtcact aaattttgca atggaaaacg gcgataaaga agacagcgac 2280

aatgaatag 2289

<210> 8

<211> 1866

<212> DNA

<213> Artificial Sequence

<220>

<223> Rep gene

<400> 8

ctggcggggt tttacgagat tgtgattaag gtccccagcg accttgacgg gcatctgccc 60

ggcatttctg acagctttgt gaactgggtg gccgagaagg agtgggagtt gccgccagat 120

tctgacttgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180

cgcgactttc tgacggagtg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 240

caatttgaga agggagagag ctacttccac ttacacgtgc tcgtggaaac caccggggtg 300

aaatccttag ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaacggc 420

gccggaggcg ggaacaaggt ggtggacgag tgctacatcc ccaattactt gctccccaaa 480

acccagcctg agctccagtg ggcgtggact aatttagaac agtatttaag cgcctgtttg 540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600

gagcagaaca aagagaatca gaatcccaat tctgacgcgc cggtgatcag atcaaaaact 660

tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 720

cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 780

tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 840

cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 900

attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 960

acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1020

accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1080

aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1140

aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1200

gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1260

aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1320

ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1380

gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1440

gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1500

gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1560

gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1620

aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1680

ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1740

tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1800

ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1860

caataa 1866

<210> 9

<211> 2211

<212> DNA

<213> Artificial Sequence

<220>

<223> Cap gene

<400> 9

ctggctgccg acggttatct acccgattgg ctcgaggaca accttagtga aggaattcgc 60

gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120

aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180

aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660

gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840

tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900

ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960

caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020

acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080

gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140

acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200

ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260

cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320

gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380

ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440

ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500

tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560

ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620

ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680

accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740

gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 1800

atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 1860

aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 1920

aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 1980

gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 2040

gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 2100

tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 2160

tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgta a 2211

<210> 10

<211> 5623

<212> DNA

<213> Artificial Sequence

<220>

<223> Ac96-Rep-CNE-Cap

<400> 10

tcaaaaaaaa ttgtaaaatg ttgtcaatca tgttggctat cgtgtttgta cttttcgtgt 60

taatttattt aataatttcg atcaaaaatc accatccatt cttacataga atagaaacgc 120

taatacaaga tttcaacaac acattgttgt ttggcgcgta tgtacagatt tacgatttaa 180

gcacgcccgc ccgcaccgaa cgattgttta ttattgcgcc cgaaaatgtg gtgttgtata 240

attttaacaa aacgctctat tattacttgg actcggcgaa cgtgttttgt cccaacgagt 300

ttagcgtgac cacgttcacg caatccacta ttaaaacgat caacgagacg ggaatatatg 360

ccaccgcatg cacgccggtc agcagcttga cgctaattga acattttgca acattaaaaa 420

ataacgtgcc cgatcacacg ctcgttctcg gaacaaacga cccaacaccc gtgcgtttta 480

ttctgtcttt ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt 540

tcagttagcc tcccccatct cccggtacct tattgttcaa agatgcagtc atccaaatcc 600

acattgacca gatcgcaggc agtgcaagcg tctggcacct ttcccatgat atgatgaatg 660

tagcacagtt tctgatacgc ctttttgacg acagaaacgg gttgagattc tgacacggga 720

aagcactcta aacagtcttt ctgtccgtga gtgaagcaga tatttgaatt ctgattcatt 780

ctctcgcatt gtctgcaggg aaacagcatc agattcatgc ccacgtgacg agaacatttg 840

ttttggtacc tgtctgcgta gttgatcgaa gcttccgcgt ctgacgtcga tggctgcgca 900

actgactcgc gcacccgttt gggctcactt atatctgcgt cactgggggc gggtcttttc 960

ttggctccac cctttttgac gtagaattca tgctccacct caaccacgtg atcctttgcc 1020

caccggaaaa agtctttgac ttcctgcttg gtgaccttcc caaagtcatg atccagacgg 1080

cgggtgagtt caaatttgaa catccggtct tgcaacggct gctggtgttc gaaggtcgtt 1140

gagttcccgt caatcacggc gcacatgttg gtgttggagg tgacgatcac gggagtcggg 1200

tctatctggg ccgaggactt gcatttctgg tccacgcgca ccttgcttcc tccgagaatg 1260

gctttggccg actccacgac cttggcggtc atcttcccct cctcccacca gatcaccatc 1320

ttgtcgacac agtcgttgaa gggaaagttc tcattggtcc agtttacgca cccgtagaag 1380

ggcacagtgt gggctatggc ctccgcgatg ttggtcttcc cggtagttgc aggcccaaac 1440

agccagatgg tgttcctctt gccgaacttt ttcgtggccc atcccagaaa gacggaagcc 1500

gcatattggg gatcgtaccc gtttagttcc aaaattttat aaatccgatt gctggaaatg 1560

tcctccacgg gctgctggcc caccaggtag tcgggggcgg ttttagtcag gctcataatc 1620

tttcccgcat tgtccaaggc agccttgatt tgggaccgcg agttggaggc cgcattgaag 1680

gagatgtatg aggcctggtc ctcctggatc cactgcttct ccgaggtaat ccccttgtcc 1740

acgagccacc cgaccagctc catgtacctg gctgaagttt ttgatctgat caccggcgcg 1800

tcagaattgg gattctgatt ctctttgttc tgctcctgcg tctgcgacac gtgcgtcaga 1860

tgctgcgcca ccaaccgttt acgctccgtg agattcaaac aggcgcttaa atactgttct 1920

aaattagtcc acgcccactg gagctcaggc tgggttttgg ggagcaagta attggggatg 1980

tagcactcgt ccaccacctt gttcccgcct ccggcgccgt ttctggtctt tgtgaccgcg 2040

aaccagtttg gcaaagtcgg ctcgatcccg cggtaaattc tctgaatcag tttttcgcga 2100

atctgactca ggaaacgtcc caaaactaag gatttcaccc cggtggtttc cacgagcacg 2160

tgtaagtgga agtagctctc tcccttctca aattgcacaa agaaaagggc ctccggggcc 2220

ttactcacac ggcgccactc cgtcagaaag tcgcgctgca gcttctcggc cacggtcagg 2280

ggtgcctgct caatcagatt cagatccaag tcagaatctg gcggcaactc ccactccttc 2340

tcggccaccc agttcacaaa gctgtcagaa atgccgggca gatgcccgtc aaggtcgctg 2400

gggaccttaa tcacaatctc gtaaaacccc gccagggcgg ccccgggtga tcaagtcttc 2460

gtcgagtgat tgtaaataaa atgtaattta cagtatagta ttttaattaa tatacaaatg 2520

atttgataat aattcttatt taactataat atattgtgtt gggttgaatt aaaggtccgt 2580

atacactttt ttgtaatgca aaaaagttga tagtgtagta gtatattggg agcgtatcgt 2640

acagtgtaga ctattctaat aaaatagtct acgatttgta gagattgtac tgtatatgga 2700

gtgtcaggca aaagtgaact tttttgcatt gcaaaaaaat tccggaatat taatagatca 2760

tggagataat taaaatgata accatctcgc aaataaataa gtattttact gttttcgtaa 2820

cagttttgta ataaaaaaac ctataaatat tccggattat tcataccgtc ccaccatcgg 2880

gcgcggatcc gccgccctgg ctgccgacgg ttatctaccc gattggctcg aggacaacct 2940

tagtgaagga attcgcgagt ggtgggcttt gaaacctgga gcccctcaac ccaaggcaaa 3000

tcaacaacat caagacaacg ctcgaggtct tgtgcttccg ggttacaaat accttggacc 3060

cggcaacgga ctcgacaagg gggagccggt caacgcagca gacgcggcgg ccctcgagca 3120

cgacaaggcc tacgaccagc agctcaaggc cggagacaac ccgtacctca agtacaacca 3180

cgccgacgcc gagttccagg agcggctcaa agaagatacg tcttttgggg gcaacctcgg 3240

gcgagcagtc ttccaggcca aaaagaggct tcttgaacct cttggtctgg ttgaggaagc 3300

ggctaagacg gctcctggaa agaagaggcc tgtagagcag tctcctcagg aaccggactc 3360

ctccgcgggt attggcaaat cgggtgcaca gcccgctaaa aagagactca atttcggtca 3420

gactggcgac acagagtcag tcccagaccc tcaaccaatc ggagaacctc ccgcagcccc 3480

ctcaggtgtg ggatctctta caatggcttc aggtggtggc gcaccagtgg cagacaataa 3540

cgaaggtgcc gatggagtgg gtagttcctc gggaaattgg cattgcgatt cccaatggct 3600

gggggacaga gtcatcacca ccagcacccg aacctgggcc ctgcccacct acaacaatca 3660

cctctacaag caaatctcca acagcacatc tggaggatct tcaaatgaca acgcctactt 3720

cggctacagc accccctggg ggtattttga cttcaacaga ttccactgcc acttctcacc 3780

acgtgactgg cagcgactca tcaacaacaa ctggggattc cggcctaagc gactcaactt 3840

caagctcttc aacattcagg tcaaagaggt tacggacaac aatggagtca agaccatcgc 3900

caataacctt accagcacgg tccaggtctt cacggactca gactatcagc tcccgtacgt 3960

gctcgggtcg gctcacgagg gctgcctccc gccgttccca gcggacgttt tcatgattcc 4020

tcagtacggg tatctgacgc ttaatgatgg aagccaggcc gtgggtcgtt cgtcctttta 4080

ctgcctggaa tatttcccgt cgcaaatgct aagaacgggt aacaacttcc agttcagcta 4140

cgagtttgag aacgtacctt tccatagcag ctacgctcac agccaaagcc tggaccgact 4200

aatgaatcca ctcatcgacc aatacttgta ctatctctca aagactatta acggttctgg 4260

acagaatcaa caaacgctaa aattcagtgt ggccggaccc agcaacatgg ctgtccaggg 4320

aagaaactac atacctggac ccagctaccg acaacaacgt gtctcaacca ctgtgactca 4380

aaacaacaac agcgaatttg cttggcctgg agcttcttct tgggctctca atggacgtaa 4440

tagcttgatg aatcctggac ctgctatggc cagccacaaa gaaggagagg accgtttctt 4500

tcctttgtct ggatctttaa tttttggcaa acaaggaact ggaagagaca acgtggatgc 4560

ggacaaagtc atgataacca acgaagaaga aattaaaact actaacccgg tagcaacgga 4620

gtcctatgga caagtggcca caaaccacca gagtgcccaa gcacaggcgc agaccggctg 4680

ggttcaaaac caaggaatac ttccgggtat ggtttggcag gacagagatg tgtacctgca 4740

aggacccatt tgggccaaaa ttcctcacac ggacggcaac tttcaccctt ctccgctgat 4800

gggagggttt ggaatgaagc acccgcctcc tcagatcctc atcaaaaaca cacctgtacc 4860

tgcggatcct ccaacggcct tcaacaagga caagctgaac tctttcatca cccagtattc 4920

tactggccaa gtcagcgtgg agatcgagtg ggagctgcag aaggaaaaca gcaagcgctg 4980

gaacccggag atccagtaca cttccaacta ttacaagtct aataatgttg aatttgctgt 5040

taatactgaa ggtgtatata gtgaaccccg ccccattggc accagatacc tgactcgtaa 5100

tctgtaaaag cttgtcgaga agtactagag gatcataatc agccatacca catttgtaga 5160

ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa 5220

tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag 5280

catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 5340

actcatcaat gtatcttatc atgtctggat ctgatcactg cttgagccta ggatgtggtc 5400

gaccaacaga ttcagttttc aatactcgac attatcaatt atttgattta caatggctac 5460

gtggatttgt tggccgaata acgcgtatat agacgcttgt acgttcatcg tagtaatcat 5520

tttaatacat ttgattgaac taaacataca tctgcaatgg gtgaaagagt cactaaattt 5580

tgcaatggaa aacggcgata aagaagacag cgacaatgaa tag 5623

<210> 11

<211> 5667

<212> DNA

<213> Artificial Sequence

<220>

<223> Ac96-Rep-NAE-Cap

<400> 11

tcaaaaaaaa ttgtaaaatg ttgtcaatca tgttggctat cgtgtttgta cttttcgtgt 60

taatttattt aataatttcg atcaaaaatc accatccatt cttacataga atagaaacgc 120

taatacaaga tttcaacaac acattgttgt ttggcgcgta tgtacagatt tacgatttaa 180

gcacgcccgc ccgcaccgaa cgattgttta ttattgcgcc cgaaaatgtg gtgttgtata 240

attttaacaa aacgctctat tattacttgg actcggcgaa cgtgttttgt cccaacgagt 300

ttagcgtgac cacgttcacg caatccacta ttaaaacgat caacgagacg ggaatatatg 360

ccaccgcatg cacgccggtc agcagcttga cgctaattga acattttgca acattaaaaa 420

ataacgtgcc cgatcacacg ctcgttctcg gaacaaacga cccaacaccc gtgcgtttta 480

ttctgtcttt ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt 540

tcagttagcc tcccccatct cccggtacct tattgttcaa agatgcagtc atccaaatcc 600

acattgacca gatcgcaggc agtgcaagcg tctggcacct ttcccatgat atgatgaatg 660

tagcacagtt tctgatacgc ctttttgacg acagaaacgg gttgagattc tgacacggga 720

aagcactcta aacagtcttt ctgtccgtga gtgaagcaga tatttgaatt ctgattcatt 780

ctctcgcatt gtctgcaggg aaacagcatc agattcatgc ccacgtgacg agaacatttg 840

ttttggtacc tgtctgcgta gttgatcgaa gcttccgcgt ctgacgtcga tggctgcgca 900

actgactcgc gcacccgttt gggctcactt atatctgcgt cactgggggc gggtcttttc 960

ttggctccac cctttttgac gtagaattca tgctccacct caaccacgtg atcctttgcc 1020

caccggaaaa agtctttgac ttcctgcttg gtgaccttcc caaagtcatg atccagacgg 1080

cgggtgagtt caaatttgaa catccggtct tgcaacggct gctggtgttc gaaggtcgtt 1140

gagttcccgt caatcacggc gcacatgttg gtgttggagg tgacgatcac gggagtcggg 1200

tctatctggg ccgaggactt gcatttctgg tccacgcgca ccttgcttcc tccgagaatg 1260

gctttggccg actccacgac cttggcggtc atcttcccct cctcccacca gatcaccatc 1320

ttgtcgacac agtcgttgaa gggaaagttc tcattggtcc agtttacgca cccgtagaag 1380

ggcacagtgt gggctatggc ctccgcgatg ttggtcttcc cggtagttgc aggcccaaac 1440

agccagatgg tgttcctctt gccgaacttt ttcgtggccc atcccagaaa gacggaagcc 1500

gcatattggg gatcgtaccc gtttagttcc aaaattttat aaatccgatt gctggaaatg 1560

tcctccacgg gctgctggcc caccaggtag tcgggggcgg ttttagtcag gctcataatc 1620

tttcccgcat tgtccaaggc agccttgatt tgggaccgcg agttggaggc cgcattgaag 1680

gagatgtatg aggcctggtc ctcctggatc cactgcttct ccgaggtaat ccccttgtcc 1740

acgagccacc cgaccagctc catgtacctg gctgaagttt ttgatctgat caccggcgcg 1800

tcagaattgg gattctgatt ctctttgttc tgctcctgcg tctgcgacac gtgcgtcaga 1860

tgctgcgcca ccaaccgttt acgctccgtg agattcaaac aggcgcttaa atactgttct 1920

aaattagtcc acgcccactg gagctcaggc tgggttttgg ggagcaagta attggggatg 1980

tagcactcgt ccaccacctt gttcccgcct ccggcgccgt ttctggtctt tgtgaccgcg 2040

aaccagtttg gcaaagtcgg ctcgatcccg cggtaaattc tctgaatcag tttttcgcga 2100

atctgactca ggaaacgtcc caaaactaag gatttcaccc cggtggtttc cacgagcacg 2160

tgtaagtgga agtagctctc tcccttctca aattgcacaa agaaaagggc ctccggggcc 2220

ttactcacac ggcgccactc cgtcagaaag tcgcgctgca gcttctcggc cacggtcagg 2280

ggtgcctgct caatcagatt cagatccaag tcagaatctg gcggcaactc ccactccttc 2340

tcggccaccc agttcacaaa gctgtcagaa atgccgggca gatgcccgtc aaggtcgctg 2400

gggaccttaa tcacaatctc gtaaaacccc gccagggcgg ccccgggtga tcaagtcttc 2460

gtcgagtgat tgtaaataaa atgtaattta cagtatagta ttttaattaa tatacaaatg 2520

atttgataat aattcttatt taactataat atattgtgtt gggttgaatt aaaggtccgt 2580

ataccatttt cagcgacgta tattgacaaa tatactacag tcggacgttt gtgccgacct 2640

atatactaca ctttaccaaa aatatactac actaaactct aaatatacta caactccact 2700

tcaatataac cacactctcg taaaacggcc caaaaatatc gaaatatatg gggcaaatac 2760

acgtttaaaa aacgctacga ttcctccgga atattaatag atcatggaga taattaaaat 2820

gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt tgtaataaaa 2880

aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg atccgccgcc 2940

ctggctgccg acggttatct acccgattgg ctcgaggaca accttagtga aggaattcgc 3000

gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 3060

aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 3120

aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 3180

cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 3240

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 3300

gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 3360

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 3420

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 3480

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 3540

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 3600

gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 3660

accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 3720

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 3780

tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 3840

ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 3900

caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 3960

acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 4020

gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 4080

acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 4140

ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 4200

cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 4260

gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 4320

ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 4380

ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 4440

tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 4500

ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 4560

ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 4620

accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 4680

gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 4740

atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 4800

aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 4860

aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 4920

gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 4980

gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 5040

tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 5100

tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgta aaagcttgtc 5160

gagaagtact agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta 5220

aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 5280

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 5340

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 5400

tatcatgtct ggatctgatc actgcttgag cctaggatgt ggtcgaccaa cagattcagt 5460

tttcaatact cgacattatc aattatttga tttacaatgg ctacgtggat ttgttggccg 5520

aataacgcgt atatagacgc ttgtacgttc atcgtagtaa tcattttaat acatttgatt 5580

gaactaaaca tacatctgca atgggtgaaa gagtcactaa attttgcaat ggaaaacggc 5640

gataaagaag acagcgacaa tgaatag 5667

<210> 12

<211> 2407

<212> DNA

<213> Artificial Sequence

<220>

<223> ITR-GOI

<400> 12

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggccgca cgcgccgccc gtcagtgggc agagcgcaca 180

tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga 240

aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag 300

ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg 360

tttgccgcca gaacacgcgt aagggatccg ccaccatggt gagcaagggc gaggaggata 420

acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg 480

gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg 540

ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc 600

agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga 660

agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg 720

tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc 780

tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg 840

aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga 900

ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca 960

agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc 1020

acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg 1080

gcggcatgga cgagctgtac aagtaagaat tcgatatcaa gcttatcgat aatcaacctc 1140

tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc 1200

tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca 1260

ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg 1320

tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca 1380

ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg 1440

cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg 1500

acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg 1560

ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg 1620

accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc 1680

ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg 1740

agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt 1800

tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga 1860

ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt 1920

tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc 1980

acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc 2040

tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg 2100

gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat 2160

ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc 2220

tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg 2280

atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 2340

gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 2400

ctgcagg 2407

Claims (18)

1. A composition for producing a recombinant baculovirus in insect cells, comprising: comprising a packaging-deficient baculovirus plasmid that lacks a CNE sequence and/or a NAE sequence in a baculovirus genome and a first rescue recombinant DNA comprising a first insertion sequence comprising a first functional fragment and a first complementing sequence that is at least one of the sequences deleted from the packaging-deficient baculovirus plasmid and a first homology arm flanking the first insertion sequence, said composition being capable of undergoing homologous recombination in insect cells to produce a recombinant baculovirus.

2. The composition of claim 1, wherein: the first functional fragment is a cap gene expression cassette of AAV and a rep gene expression cassette of AAV.

3. The composition of claim 2, wherein: the first insertion sequence comprises, in order from 5 'to 3', the cap gene expression cassette, the first complementing sequence and the rep gene expression cassette.

4. The composition of claim 2, wherein: the first insertion sequence comprises, in order from 5 'to 3', the rep gene expression cassette, the first complementing sequence and the cap gene expression cassette.

5. The composition of claim 2, wherein: the first complementing sequence is positioned between the cap gene expression cassette and the rep gene expression cassette, and two ends of the first complementing sequence are respectively close to the initiating end of the cap gene expression cassette and the initiating end of the rep gene expression cassette.

6. The composition of claim 2, wherein: the packaging-defective baculovirus plasmid is a recombinant bacmid comprising an AAV ITR core expression element with a foreign gene.

7. The composition of claim 6, wherein: the recombinant bacmid was obtained by a baculovirus transfer vector mediated transposition of Tn 7.

8. The composition of claim 1, wherein: the first functional fragment is an AAV ITR core expression element with an exogenous gene.

9. The composition of claim 8, wherein: the packaging defect type baculovirus plasmid is a recombinant bacmid containing an AAV cap gene expression cassette and an AAV rep gene expression cassette.

10. The composition of claim 9, wherein: the recombinant bacmid was obtained by baculovirus transfer vector mediated transposition of Tn 7.

11. The composition of claim 1, wherein: the first functional fragment is a reporter gene.

12. The composition of claim 1, wherein: the first functional fragment is a nucleotide sequence encoding a therapeutic gene product.

13. The composition of claim 1, wherein: also included is a second rescue recombinant DNA that lacks a CNE sequence and a NAE sequence in the baculovirus genome, the second rescue recombinant DNA comprising a second insertion sequence comprising a second complementing sequence, the first complementing sequence being a CNE sequence or a NAE sequence, and a second homology arm flanking the second insertion sequence, the second complementing sequence being a sequence that is different from the first complementing sequence in the CNE sequence and the NAE sequence.

14. The composition of claim 13, wherein: the first functional fragment is a cap gene expression box of AAV and a rep gene expression box of AAV, the second insertion sequence also comprises a second functional fragment, and the second functional fragment is an AAV ITR core expression element with exogenous genes.

15. Use of a composition according to any one of claims 1 to 14 for the preparation of recombinant baculovirus and/or recombinant adeno-associated virus in insect cells.

16. An insect cell, characterized by: comprising the composition of any one of claims 1-14.

17. A method of growing or producing recombinant baculovirus in vitro, comprising: comprising co-transfecting an insect cell with the composition of any one of claims 1-14 and culturing the insect cell.

18. A method of growing or producing recombinant adeno-associated virus in vitro, comprising: comprising co-transfecting an insect cell with the composition of any one of claims 6, 7, 9, 10 and 14 and culturing the insect cell.

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