CN117925707A - Infectious clone virus of virulent strain of NADC30-like porcine reproductive and respiratory syndrome virus and application thereof - Google Patents
Infectious clone virus of virulent strain of NADC30-like porcine reproductive and respiratory syndrome virus and application thereof Download PDFInfo
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Abstract
The invention discloses an infectious clone plasmid pACYC177-rSD17-38 and a construction method thereof, and also discloses an infectious clone virus and a construction method thereof, and the invention also discloses an NADC30-LIKE PRRSV modified strain which can adapt to Marc-145 cell in-vitro culture. The invention also discloses an infectious clone plasmid pACYC177-rSD17-38, the infectious clone virus and the application of the modified strain in preparing vaccines of porcine reproductive and respiratory syndrome virus. The modified virus constructed by the invention can be inoculated into Marc-145 passage cells to cause cytopathy. The reverse genetic operation platform and Marc-145 adapted modified virus constructed by the invention can be used for developing the first NADC30-LIKE PRRSV genetic engineering vaccine in China, and is beneficial to the prevention and control of PRRS epidemic situation in China.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a NADC30-like porcine reproductive and respiratory syndrome virus virulent strain infectious clone virus and application thereof, in particular to a Marc-145 cell-adapted chimeric virus obtained by rescue and transformation.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the important infectious diseases causing abortion in sows and respiratory syndrome in piglets, which causes huge economic losses to the pig industry worldwide. The etiology of PRRS is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). PRRSV can be divided into two species (species): PRRSV1 and PRRSV2. In recent years, PRRSV2 is popular in our country. HP-PRRSV, NADC30-LIKE PRRSV and NADC34-LIKE PRRSV are the main. The NADC30-LIKE PRRSV is strong in toxicity and infectivity, popular in a plurality of provinces and cities in China, is a strain with highest current popularity in China, and causes serious harm to healthy cultivation of live pigs in China.
PRRSV2 belongs to the family arteriviridae and is a single positive strand RNA virus with a genome size of about 15kb. Comprising at least 10 Open Reading Frames (ORFs). The ORF1a and ORF1b genes encode at least 16 nonstructural proteins and the ORF2-7 gene encodes 8 structural proteins. Among them, the three small envelope proteins (GP 2a, GP3 and GP 4) encoded by the ORF2-4 gene play a key role in determining the cell tropism of PRRSV strains.
Vaccine immunization is an effective means for controlling PRRS epidemic situation. However, commercial PRRSV attenuated vaccines (JXA 1-R, R98, huN-F112 and the like) in China have poor cross protection effect on NADC30-LIKE PRRSV. Furthermore, the Marc-145 cell line is a passaged cell line for PRRSV vaccine production, whereas the NADC30-LIKE PRRSV wild strain is not adapted to Marc-145 cells, which is one of the major obstacles impeding NADC30-like vaccine development. Therefore, there is an urgent need to develop a NADC30-LIKE PRRSV vaccine strain which can adapt to the in vitro subculture of Marc-145 cell line.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing an infectious clone plasmid pACYC177-rSD17-38 and a construction method thereof.
The invention aims to solve the technical problem of providing an infectious clone virus and a construction method thereof.
The invention also solves the technical problem of providing an NADC30-LIKE PRRSV modified strain which can adapt to Marc-145 cell in-vitro culture.
The invention also solves the technical problem of providing the application of the infectious clone plasmid pACYC177-rSD17-38, the infectious clone virus or the modified strain in preparing vaccines of porcine reproductive and respiratory syndrome virus.
The invention finally solves the technical problem of providing a vaccine for porcine reproductive and respiratory syndrome virus.
The technical scheme is as follows: in order to solve the technical problems, the invention provides an infectious clone plasmid which is obtained by connecting an intermediate vector with a gene fragment F4, wherein the intermediate vector is obtained by connecting gene fragments F1, F2 and F3 with a plasmid pACYC177 in a homologous recombination mode, and the gene fragments F1, F2, F3 and F4 are sequentially positioned at nucleotide numbers 1-1525, nucleotide numbers 1481-7891, nucleotide numbers 7844-12371 and nucleotide numbers 12321-15177 of the whole genome of the NADC30-LIKE PRRSV SD-38 virus.
The invention also discloses a construction method of the infectious clone plasmid pACYC177-rSD17-38, which comprises the following steps:
1) Respectively designing primer pairs of gene fragments F1, F2 and F3, and then respectively amplifying to obtain SD17-38-F1, SD17-38-F2 and SD17-38-F3 fragments;
2) Designing a primer pair of a gene fragment F4, firstly using a primer pair of SD17-38-Bsp1407IF4 and SD17-38-4R1 to amplify to obtain F4-1, then using F4-1 as a template, adding a primer SD17-38-AscI-4R2 of a gene sequence (SEQ ID NO. 1) of hepatitis delta virus into the 3' -end of the primer SD17-38-Bsp1407IF4, and amplifying to obtain F4;
3) The pACYC177 plasmid is subjected to double enzyme tangential treatment by PacI and Bsp1407I, F1, F2 and F3 fragments are subjected to homologous recombination with the linearization vector to obtain plasmid pACYC177-rSD17-38-F1+F2+F3 connected with the F1, F2 and F3 fragments, then pACYC177-rSD17-38-F1+F2+F3 plasmid and F4 fragment are subjected to double enzyme digestion by Bsp1407I and AscI, and then F4 fragment and the vector are subjected to enzyme digestion connection to obtain the complete pACYC177-rSD17-38 plasmid.
Wherein the homologous recombination system comprises 20-40 ng of F1 fragment, 100-200 ng of F2 fragment, 50-150 ng of F3 fragment and 100-300 ng of pACYC177 linearization vector, and 1 mu L Exanse MultiS and 5X CE MultiS Buffuer mu L of pACYC177 linearization vector are added for 30min at 37 ℃.
The invention also comprises an infectious clone virus, wherein the infectious clone is obtained by transfecting cells with the plasmid and then infecting susceptible cells.
The invention also discloses a rescue method of the infectious clone virus, which comprises the following steps: and (3) transfecting the pACYC177-rSD17-38 plasmid into BHK-21 cells, inoculating the primary PAM cells into the obtained transfection solution, and culturing for several days to obtain the rescued infectious clone virus.
The invention also comprises an NADC30-LIKE PRRSV modified strain which can adapt to the in vitro culture of Marc-145 cells, and is characterized in that the modified strain is obtained by replacing part of the coding gene of the small envelope protein of the infectious clone virus with the part of the coding gene of the small envelope protein of the HP-PRRSV XJ17-5 isolate.
Wherein, the partial coding genes of the replaced small envelope proteins comprise coding genes of amino acids 1-191 of XJ17-5 strain ORF2-ORF3-ORF4 or XJ17-5 strain ORF2, the gene sequence of the XJ17-5 strain ORF2-ORF3-ORF4 is shown as nucleotide 1-1705 of SEQ ID NO.2, and the gene sequence of the XJ17-5 strain ORF2 is shown as nucleotide 1-573bp of SEQ ID NO. 2.
The invention also comprises the application of the infectious clone plasmid pACYC177-rSD17-38, the infectious clone virus or the modified strain in preparing vaccines of porcine reproductive and respiratory syndrome virus.
The invention also includes a vaccine for porcine reproductive and respiratory syndrome virus, which comprises the infectious clone virus or the engineered strain.
In summary, the invention provides a reverse genetic operating platform of NADC30-LIKE PRRSV and a construction method thereof. Specifically, pACY plasmid is used as a carrier, and the whole gene component of the NADC30-LIKE PRRSV SD17-38 virulent strain is connected to the carrier in a segmented way by utilizing methods of PCR amplification, enzyme digestion connection, homologous recombination and the like to obtain SD17-38 full-length infectious clone, so that the infectious clone virus rSD17-38 is constructed and saved, and the infectious clone platform of the first NADC30-LIKE PRRSV virulent strain is successfully constructed.
In another aspect of the invention, methods are provided for engineering an adaptable Marc-145 cell subculture NADC30-LIKE PRRSV strain. Specifically comprises the steps of replacing the sequence of the small envelope protein synthesis gene ORF2-ORF3-ORF4 of the Marc-145 cell adaptation strain XJ17-5 and the sequence of GP2a-1-191aa to corresponding fragments in the infectious clone of NADC30-LIKE PRRSV by using methods such as homologous recombination and the like based on a reverse genetic operation platform of NADC30-LIKE PRRSV in the invention, and constructing an NADC30-LIKE PRRSV modified strain which can save two strains of the adaptable Marc-145 cells.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
1. The invention uses NADC30-LIKE PRRSV virulent strain SD17-38 as parent virus to construct reverse genetic operation platform-infectious clone plasmid pACYC177-rSD17-38;
2. The infectious clone virus obtained by the invention has good proliferation efficacy on Primary Alveolar Macrophages (PAM);
3. two NADC30-LIKE PRRSV modified viruses which can adapt to Marc-145 are obtained based on NADC30-LIKE PRRSV reverse genetic operation platform modification;
4. The engineered virus constructed by the invention can be inoculated into Marc-145 passage cells to cause Cytopathy (CPE).
5. The reverse genetic operation platform and Marc-145 adapted modified virus constructed by the invention can be used for developing the first NADC30-LIKE PRRSV genetic engineering vaccine in China, and is beneficial to the prevention and control of PRRS epidemic situation in China.
Drawings
FIG. 1 is a sequence alignment chart of SD17-36 and SD17-38 in example 1;
FIG. 2 is a schematic diagram of the construction strategy of SD17-36 and SD17-38 infectious clones in example 1;
FIG. 3 is a diagram showing the results of PCR amplification of 4 fragments of the SD17-36 and SD17-38 genomes in example 1;
FIG. 4 is an indirect immunofluorescence assay of example 1 for detection of rescued SD17-36 and rSD17-38 using the indirect immunofluorescence method of monoclonal antibody 6A1 against PRRSV-N protein;
FIG. 5 is the result of the fluorescent quantitative PCR method for detecting the dynamic proliferation of SD17-38 and its infectious clone virus rSD17-38 rescued on primary PAM cells in example 1;
FIG. 6 is a schematic representation of the construction strategy of the rSTX, rSTX, 234, rSTX, 34 and rSTX2-1-191aa chimeric viruses of example 2;
FIG. 7 is the results of indirect immunofluorescence assays of the rSTX, rSTX, 234, rSTX, and rSTX2-1-191aa chimeric viruses of example 2 on Marc-145 cells at 6dpi using monoclonal antibody 6A1 directed against PRRSV-N protein on rSTX234 and rSTX2-1-191aa chimeric viruses replicated on Marc-145 cells;
FIG. 8 is the dynamic proliferation results of rSTX, rSTX, 234, rSTX, 34 and rSTX2-1-191aa chimeric viruses on Marc-145 cells in example 2;
FIG. 9 is a plaque plot of rSTX, rSTX, 234, rSTX, 34 and rSTX2-1-191aa chimeric viruses on Marc-145 cells in example 2.
Detailed Description
For routine experimentation in the following examples, see the third edition of the molecular cloning Experimental guidelines written by Sambrook et al (Beijing: science Press, 2002), the use of the apparatus is referred to the instruction manual of the apparatus.
In the examples of the present invention, the virus was NADC30-LIKE PRRSV SD17-36 strain isolated in the laboratory (SD 17-36 for short), the isolated disease was lung tissue disease material from Shandong province, genBank accession No. MH 121061), NADC30-LIKE PRRSV SD-38 virulent isolate isolated in the laboratory (SD 17-38 for short), isolated disease material was lung tissue disease material from Shandong province, genBank accession No. MH 068878), HP-PRRSVXJ 17-5 isolate isolated in the laboratory (isolated disease material was lung tissue disease material from Sinkiang Uygur autonomous region, genBank accession No. MK 759853) and infectious gram Long Du rXJ-5 constructed in the laboratory and infectious clone plasmid pAC 177-rXJ-5. Cells include the BHK-21 and Marc-145 cell lines subcultured in this laboratory, and primary alveolar macrophage PAM. PRRSV anti-N protein monoclonal antibody 15A1 and PRRSV anti-N protein monoclonal antibody 6A1 are both complimentary to the professor Li Xiangdong, university of dulcimer. (all of the above materials are disclosed).
In the examples of the present invention, plasmids and strains: pACYC177 plasmid was purchased from Guangzhou from Youbao biotechnology Co., ltd, and Trans1-T1 competent cells (CD 501-02) were purchased from Beijing full gold biosystems Co.
In embodiments of the invention, other reagents are used: RNASE FREE H 2 O (R1600) was purchased from Solarbio, and pancreatin cell digestate (S310 JV) (phenol red) was purchased from Solarbio; TRIpure Reagent Total RNA extraction reagent was purchased from Edley biosystems; PRIMESCRIPT1ST STRAND CDNA SYNTHESIS KIT (6110A), 2X PRIMESTAR MAX DNA POLYMEARSE (R045) were purchased from TAKARA company; fastPure PLASMID MINI KIT (DC 201-01) was purchased from Novain Biotech Co., ltd; homologous recombination reagent (C115-01) was purchased from Novain Biotechnology Co., ltd; DMEM medium (SH 30243.01) was purchased from HyClone biochemicals ltd; fetal bovine serum (FBSKM 0503) was purchased from australian company; dylight 594, coat anti-Mouse IgG (H+L) Secondary Antibody from Invitrogen; DNA MARKER available from Zhejiang Bogijin technologies, inc.; gel Extraction Kit (CW 2302M) was purchased from Beijing as century Biotech Co., ltd; DNA restriction endonucleases were purchased from Thermo Fisher company; lipofectamine TM 3000Transfection Reagentl (L3000015) was purchased from Invitrogen corporation; opti-MEM (31985070) was purchased from Gibco corporation.
EXAMPLE 1 construction of NADC30-LIKE PRRSV SD17-36 and SD17-38 reverse genetic manipulation platforms
NADC30-LIKE PRRSV SD17-36 and SD17-38 strains were aligned for full length using the website (https:// benchling. Com /). As shown in FIG. 1, the homology of the two NADC30-LIKE PRRSV strains is only 93.13%. Therefore, different construction strategies are designed for the two strains, and the construction method is as follows.
1. Primer design for reverse genetic operating system construction
According to the construction strategy of the two strains shown in FIG. 2, the positions of restriction enzyme sites on the viral genome were selected, and primers for amplifying each fragment of NADC30-LIKE PRRSV SD strain 17-36 and SD17-38 strain were designed using Primer 5.0. The advantages are as follows: firstly, constructing infectious clone plasmids by two methods of homologous recombination and enzyme digestion connection, and continuously completing the infectious clone construction by the traditional enzyme digestion connection method when the efficiency of the homologous recombination method is low; second, the transformation of the fragments during the application of the post-infectious cloning plasmid can be facilitated. A PacI single cleavage site is introduced at the 5 'end of the genome of the NADC30-LIKE PRRSV strain, and a hepatitis delta virus (HDV Ribozyme) sequence (SEQ ID NO. 1) is added at the 3' end. The specific information of the primers is shown in tables 1 and 2.
TABLE 1 construction primers for SD17-36 infectious clone
TABLE 2 construction primers for SD17-38 infectious clones
2. Construction of SD17-36 and SD17-38 infectious clones
1) Segmented amplification of all-gene fragments of SD17-36 and SD17-38
First, 200. Mu.l of SD17-36 and SD17-38 virus stock solutions were subjected to total RNA extraction using TRIpure Reagent (Edley), and the total RNA thus extracted was stored at-20℃or at-80℃for a long period of time. The viral RNA was reverse transcribed into cDNA using a reverse transcription kit (PRIMESCRIPTTM, ST STRAND CDNA SYNTHESIS KIT, TAKARA) and the resulting cDNA was stored at-20℃for use. The reaction system and procedure are detailed in tables 3 and 4.
Table 3, SD17-36 and SD17-38 Total RNA pretreatment System and procedure
Table 4, SD17-36 and SD17-38 Total RNA reverse transcription System and procedure
As shown in FIG. 2, the SD17-36 whole genome was divided into four fragments F1 (genome 1-2329 bp), F2 (genome 2294-8740 bp), F3 (genome 8709-14052 bp) and F4 (genome 14017-15147 bp). First, SD17-36 cDNA obtained by the above-mentioned reverse transcription was used as a template. The F1 fragment was amplified by combining the upstream and downstream primer pairs SD17-36-PacI-F1 and SD17-36-XhoI-R1 in Table 1; SD17-36-XhoI-F2 and SD17-36-AflII-R2 in combination, the F2 fragment was amplified; SD17-36-AflII-F3 and SD17-36-Bsp1407I-R3, amplified F3; next, to add the Poly-A tail to the whole viral genome, the Poly-A tail sequence was synthesized into primer SD17-36-4R1, and then the first amplification was performed with the primer and SD17-36-Bsp1407I-F3 primer combination using SD17-38 cDNA as a template to obtain F4-1 (14017-15078 bp). Then, in order to add a hepatitis delta virus (HDV Ribozyme) sequence (SEQ ID NO. 1) to the 3' -end of the viral genome, the sequence is synthesized into a primer SD17-36-AscI-4R2, and then the F4-1 fragment is used as a template, and the primer and the SD17-36-Bsp1407IF3 primer are combined for second amplification to obtain F4.
The SD17-38 whole genome is divided into four fragments F1 (genome 1-1525 bp), F2 (genome 1481-7891 bp), F3 (genome 7844-12371 bp) and F4 (genome 12321-15177 bp). First, SD17-38 cDNA obtained by the above-mentioned reverse transcription was used as a template. The upstream and downstream primer pairs SD17-38-PacI-F1 and SD17-38-ScaI-R1 in Table 2; SD17-38-ScaI-F2 and SD17-38-NotI-R2; SD17-38-NotI-F3 and SD17-38-Bsp1407I-R3 were combined and amplified to obtain F1, F2 and F3 fragments, respectively. Similarly, the amplification of F4 was performed in two steps, and the combination of SD17-38-Bsp1407I-F3 and primer SD17-36-4R1 was amplified for the first time to give F4-1 (12321-15113 bp). Then, the primer SD17-38-Bsp1407I-F3 and the primer SD17-38-AscI-4R2 are combined, and the F4-1 fragment is used as a template for secondary amplification to obtain F4 (12321-15177 bp). The specific reaction system and the reaction procedure are shown in tables 5 and 6.
TABLE 5 reaction System
TABLE 6 reaction procedure
After completion of the PCR reaction, all F1, F2 and F3 and 2uL F4 PCR products were electrophoresed using agarose gel at a concentration of 0.9%. The electrophoresis result is shown in FIG. 3, and the obtained SD17-36 gene fragment F1 has the size of 2329bp, F2 has the size of 6446bp, F3 has the size of 5343 and F4 has the size of 1130bp; the obtained SD17-38 gene fragment F1 has the size of 1525bp, F2 has the size of 6410bp, F3 has the size of 4527bp and F4 has the size of 2856bp.
2) Each of the fragments of SD17-36 and SD17-38 were ligated
The amplification product was recovered using a product gel recovery kit. And then the pACYC177 plasmid is subjected to double enzyme digestion, and the gel recovery products are subjected to homologous recombination (or connection) to the pACYC177 vector in sequence, wherein the construction strategy is shown in figure 1. The specific operation steps are as follows:
First, pacI and NotI are used for double digestion of pACYC177 plasmid, and the pACYC177 digestion system is as follows: pACYC177 plasmid (200 ng/. Mu.L) 15. Mu.L, pacI 3. Mu.L, bsp1407I 3. Mu.L, 10X CutSmart Buffer. Mu.L, RNASE FREE H 2 O was added to 40. Mu.L. Reaction conditions: water bath at 37 ℃ for 30 minutes. Specific information is shown in table 8.
Table 8, pACYC177 plasmid double enzyme cutting system
| Name of the name | PACYC177 plasmid | PacI | Bsp1407I | 10×CutSmart Buffer | RNase Free H2O |
| Dosage of | 3000ng | 3μL | 3μL | 4μL | Is added to 40 mu L |
The enzyme-cut bands were separated by agarose gel electrophoresis, and the target band was cut off for gel recovery. The gel recovered linear vector pACYC177 was subjected to homologous recombination with the F1, F2 and F3 gel recovered products, the specific systems are shown in Table 9 and Table 10, and the reaction procedure was 30min at 37 ℃. The resulting products were designated pACYC177-rSD17-36-F1+F2+F3 and pACYC177-rSD17-38-F1+F2+F3.
Table 9, SD17-36 homologous recombination System
Table 10, SD17-38 homologous recombination System
The homologous recombination products were transformed into Trans1-T1 competent cells. 6 colonies are picked up and cultured overnight for enrichment, plasmid DNA is extracted, pacI and Bsp1407I are used for double enzyme digestion identification, 1% agarose gel electrophoresis is carried out, plasmids with correct enzyme digestion size are picked up for sequencing verification, and then F4 fragment connection is continued.
First, the F4 fragment was purified by PCR, and the procedure was as described in Fast Pure PLASMID MINI KIT. The pACYC177-rSD17-36-F1+F2+F3 and pACYC177-rSD17-38-F1+F2+F3 plasmids and the purified F4 fragments of SD17-36 and SD17-38 were subjected to double cleavage with Bsp1407I and AscI, and the double cleavage systems are shown in tables 11 and 12. The resulting products were pACYC177-rSD17-36-F1+F2+F3 and pACYC177-rSD17-38-F1+F2+F3 linearized vectors and F4 fragments of SD17-36 and SD17-38 with cohesive ends. Then, T4 ligase ligation was performed according to the construction strategy diagram shown in FIG. 2, and the ligation system was as shown in Table 13. Transformation and colony double enzyme digestion screening are carried out by the method, and pACYC177-rSD17-36 is sequenced by primers SD17-36-PacI-F1, SD17-36-XhoI-F2, SD17-36-AflII-F3 and SD17-38-Bsp1407I-F4 in the table 1; pACYC177-rSD17-38 was sequenced by primers SD17-38-PacI-F1, SD17-38-ScaI-F2, SD17-38-NotI-F3 and SD17-38-Bsp1407I-F3 in Table 2. The infectious clone plasmids pACYC177-rSD17-36 and pACYC177-rSD17-38 containing the whole genome sequence are finally obtained.
Table 11, pACYC177-rSD17-36-F1+F2+F3 and pACYC177-rSD17-38-F1+F2+F3 plasmid double enzyme digestion System
| Name of the name | Plasmid(s) | Bsp1407I | AscI | 10×CutSmart Buffer | RNase Free H2O |
| Dosage of | 3000ng | 3μL | 3μL | 4μL | Is added to 40 mu L |
Table 12, SD17-36 and SD 17-38F 4 fragment double cleavage System
| Name of the name | F4 fragment | Bsp1407I | AscI | 10×CutSmart Buffer | RNase Free H2O |
| Dosage of | 2000ng | 2μL | 2μL | 4μL | Is added to 40 mu L |
Table 13, T4 connection system
1.3 Rescue of rSD17-36 and rSD17-38 infectious clones
BHK-21 cells were inoculated into a 12-well cell culture plate at a density of 1X 10 6 cells/well using DMEM medium containing 10% FBS in advance, and cultured in an incubator at 37℃with 5% CO 2 until the cell density reached about 90%. According to the Lipofectamine TM 3000, 3000Transfection Reagent specification, the kit comprises two reagents, namely P3000 and Lip3000, and the cell transfection is carried out by self-provided Opti-MEM, and the specific operation is as follows:
First, a plasmid premix was prepared in an EP tube: infectious clone plasmid pACYC177-rSD17-36 or pACYC177-rSD17-38 2000ng, P3000. Mu. L, opti-MEM 50. Mu.L; in another EP tube, lip3000 premix was prepared: lip 30003. Mu.L, optin-MEM 50. Mu.L; finally, the premixed solution obtained in the two steps is mixed, and after standing for 15min at room temperature, the premixed solution is added into the BHK-21 cells. 48h after cell transfection, the 12-well plate is sealed by a sealing bag and frozen and thawed for 2 times at-80 ℃ for standby. The specific transfection system is shown in Table 14.
TABLE 14 transfection System
| Kind/name | Plasmid(s) | Lip3000 | P3000 | Opti-MEM |
| Plasmid premix | 2000ng | - | 3μL | 50μL |
| Lip3000 premix | - | 4μL | - | 50μL |
Since BHK-21 cells can only be used for transfection, they are not susceptible to PRRSV. The primary PAM cells are susceptible cells of SD17-36 and SD17-38, so BHK-21 freeze-thawing liquid inoculation is carried out by using the cells to finish virus rescue. 2mL of RPMI (1640) medium containing 2% FBS primary PAM cells were seeded at a density of 2X 10 5 cells/well on 12-well cell culture plates and incubated at 37℃with 5% CO 2. 500. Mu.L of BHK-21 cell transfection supernatant was overlaid on primary PAM cells, and after incubation for 2 hours, the supernatant was discarded, and the culture was continued by adding RPMI (1640) medium containing 2% FBS. In addition, 1X 10 3TCID50 SD17-36 and SD17-38 primary PAM cells were used as positive controls. After 3 days of culture, the cell supernatant was collected, and the cells were left for IFA identification. As shown in FIG. 4, PRRSV anti-N protein monoclonal antibody 6A1 was detected, the rescuing virus rSD17-36 did not show specific red fluorescence, while the parent virus SD17-36, rescuing virus (rSD 17-38) and parent virus (SD 17-38) showed specific red fluorescence, and the negative control group showed no specific fluorescence, confirming that rSD17-36 rescue failed, and rSD17-38 infectious cloned virus was successfully rescued on primary PAM cells.
Experimental data prove that the rescue of the infectious cloned virus of the NADC30-LIKE PRRSV strain has considerable difficulty, and only the infectious cloned virus of the SD17-38 strain is successfully rescued. The subsequent infectious clone virus engineering was performed on pACYC177-rSD17-38 plasmid.
1.4, RSD17-38 and SD17-38 proliferation Activity assays
Primary PAM cells were seeded at a density of 2×10 5 cells/well using RPMI (1640) medium containing 2ml of 2% fbs into 12-well cell culture plates, placed in an incubator at 37 ℃,5% CO 2, and after complete adherence, incubated at 1:2000 (1. Mu.L) of viral solution was added, and supernatants were collected at 2hpi (hours post infection), 24, 48, 72, 96, and TRIpure Reagent were used to extract viral RNA. mu.L of the obtained cDNA was used for real-time PCR detection of PRRSV1 ORF7 gene. The specific reaction system and amplification procedure are shown in tables 15 and 16.
TABLE 15 reaction System
| Name of the name | cDNA | 2×EX Taq | Primer pair (10 mu M) | Probe (10 mu M) | RNase Free H2O |
| Dosage of | 1μL | 20μL | 0.5 Mu L each | 0.4μL | Is added to 20 mu L |
TABLE 16 real-time PCR primer and probe sequences
| Numbering device | Primer name | Sequence (5 '-3') |
| 1 | PRRSV2-UF1 | TTGTGCTTGCTAGGCCGC |
| 2 | PRRSV2-UR1 | ACGACAAATGCGTGGTTATCA |
| 3 | Probe with a probe tip | FAM-TCTGGCCCCTGCCCA-MGB |
TABLE 17 reaction procedure
Dynamic proliferation of rSD17-38 infectious clone virus on primary PAM cells As shown in FIG. 5, rSD17-38 has good proliferation potency in vitro.
Example 2 engineering, construction and rescue of NADC30-LIKE PRRSV amenable to Marc-145 cell culture in vitro
Two infectious clones rSD17-38-T-XJ17-5-ORF5-ORF6 (rSTX) and rSD17-38-T-XJ17-5-ORF2-ORF3-ORF4 (rSTX 234) were constructed by replacing the small envelope protein (GP 2a-GP3-GP 4) encoding gene ORF2-ORF3-ORF4 and the major envelope protein (GP 5-M) encoding gene ORF5-ORF6, respectively, of the NADC30-LIKE PRRSV rSD17-38 with ORF3-ORF4 (SEQ ID NO. 2) and ORF5-ORF6 (SEQ ID NO. 3) of the in vitro culture strain HP-PRRSV XJ17-5, respectively, which are adaptable to Marc-145 cells. Experiments have found that GP2a-GP3-GP4 substitutions can adapt rSD17-38 engineered viruses to Marc-145 cells, whereas GP5-M substitutions cannot adapt rSD17-38 engineered viruses to Marc-145 cells. Two infectious clones rSD17-38-T-XJ17-5-ORF34 (rSTX 34) and rSD17-38-T-XJ17-5-ORF2a-1-191aa (rSTX-1-191 aa) were constructed by replacing the 1 st-191 aa and GP3-GP4 of GP2a of rSD17-38 with the corresponding proteins of XJ17-5, respectively. As a result rSTX-1-191 aa acquired Marc-145 cell tropism, while rSTX was unable to adapt to Marc-145 cells. Finally, we obtained two engineered strains rSTX234 and rSTX2-1-191aa that could accommodate Marc-145 cells. The specific construction process is as follows:
1. Primer design
Primers for amplifying XJ17-5 strain ORF2-ORF3-ORF4 (SEQ ID NO: 2), XJ17-5 strain ORF3-ORF4 (624-1705 bp of SEQ ID NO: 2), XJ17-5 strain ORF2-1-191aa (1-573 bp of SEQ ID NO: 2) and XJ17-5 strain ORF5-ORF6 (SEQ ID NO: 3) were designed according to the construction strategy as shown in FIG. 6 using Primer 5.0, with a homology arm of at least 20bp included between the primers. The specific information of the primers is shown in Table 18.
TABLE 18 construction primers for chimeric viruses
2. Construction and rescue of engineered viruses
The chimeric viruses rSTX, rSTX, 234, rSTX, 34 and rSTX2-1-191aa shown in FIG. 6 were constructed by substituting ORF2-ORF3-ORF4, ORF5-ORF6, ORF3-ORF4 and ORF2-1-191aa of XJ17-5 to corresponding positions of rSD17-38, respectively.
2.1 Amplification of XJ17-5-ORF5-ORF6, XJ17-5-ORF2-ORF3-ORF4, rSD17-38 front and rSD17-38 rear
Primers SD17-38-NotIF3 and rSTX-ORF 5-R in Table 18; rSTX56-ORF5-F and rSTX-ORF 6-R; rSTX56-ORF6-F and JXA1-RNOTI-2fu-1; SD17-38-NotIF3 and rSTX234-ORF2-R; rSTX-ORF 2-F and rSTX-ORF 4-R; rSTX234-ORF4-F and JXA1-RNOTI-2fu-1 are respectively combined into primer pairs which are respectively used for amplifying XJ17-5-ORF5-ORF6, rSTX front section, rSTX rear section XJ17-5-ORF2-ORF3-ORF4, rSTX front section and rSTX234 rear section, and the templates for reaction are pACYC177-rXJ17-5 plasmid and pACYC177-rSD17-38 plasmid respectively. The specific procedure of the reaction is shown in tables 19 and 20.
TABLE 19 reaction system
TABLE 20 reaction procedure
After completion of the PCR reaction, 2. Mu.L of the reaction product was taken and subjected to electrophoresis using an agarose gel having a concentration of 1%. The obtained fragment XJ17-5 strain ORF5-ORF6 has a size of 1122bp (SEQ ID NO. 3), a rSTX anterior segment of 5587bp (SEQ ID NO. 4), a rSTX posterior segment of 679bp (SEQ ID NO. 5), an XJ17-5 strain ORF2-ORF3-ORF4 has a size of 1705bp (SEQ ID NO. 2), a rSTX234 anterior segment of 3882bp (SEQ ID NO. 6) and a rSTX234 posterior segment of 1800bp (SEQ ID NO. 7).
2.2, RSTX, 234 homologous recombination of fragments
The pACYC177-rSD17-38 was double digested with NotI and AscI to obtain pACYC177-rSD17-38-F1+F2 linearized vector. The specific operation was similar to that in example 1. The specific information is shown in Table 21.
Table 21, pACYC177-rSD17-38 plasmid double enzyme digestion system
The enzyme-cut bands were separated by agarose gel electrophoresis, and the target band was cut off for gel recovery. After the gel was recovered, the concentration was measured using Nanodrop and used for further use. The XJ17-5-ORF2-ORF3-ORF4, rSTX234 front and rSTX rear were ligated into pACYC177-rSD17-38-F1+F2 linearized vectors using homologous recombination. The construction of rSTX was carried out in the same manner as rSTX234, i.e., the XJ17-5-ORF5-ORF6, rSTX56 front and rSTX rear were ligated by homologous recombination to pACYC177-rSD17-38-F1+F2 linearized vectors. The homologous recombination is specifically performed as follows:
The homologous recombination system is as follows: 77ng XJ17-5-ORF2-ORF3-ORF4, 34ng rSD17-38 front and 33ng rSD17-38 rear, 200ng pACYC177-rSD17-38-F1+F2 linearized intermediate vector, 4. Mu.l 5X CE MultiS Buffer and 2. Mu. l Exanse MultiS. The reaction conditions were 37℃for 30min. See table 22 for details.
TABLE 22 homologous recombination System
The homologous recombination products are transformed into Trans1-T1 competent cells, and independent colonies are picked for pure culture. 6 colonies were picked overnight for enrichment culture, plasmid DNA was extracted, double restriction identification was performed using NotI and AscI, 0.9% agarose gel electrophoresis, and the correct plasmid retention was identified by restriction identification and designated pACYC177-rSTX234. A positive plasmid pACYC177-rSTX was obtained in the same manner as described above.
The construction method of the plasmid pACYC177-rSTX is as follows:
After construction of the rSTX positive plasmid in 2.2 was completed, the plasmid identified correctly by double restriction enzyme was selected as a PCR template, and fragment amplification and plasmid construction of chimeric toxins rSTX and rSTX2-1-191aa were performed using the primers in Table 8, the systems and procedures in Table 9 and Table 10. The specific information is as follows: the rSTX pre-fragment was amplified using primers SD17-38-NotIF3 and rSTX34-ORF3-R, taking 1ng of the successfully rescued positive plasmid pACYC177-rSD17-38 in example 1 as template; the back end of rSTX was amplified using pACYC177-rSTX234 positive plasmid 1ng obtained in example 2.2 as a template, rSTX-ORF 3-F and the primer JXA1-R-NOTI-2 fu-1.
The construction method of plasmid pACYC177-rSTX2-1-191aa is as follows:
Taking pACYC177-rSTX234 positive plasmid 1ng as a template, and amplifying rSTX2-1-191aa front segment by using primers SD17-38-NotIF3 and rSTX2-1-191 aa-R; the post-fragment of rSTX2-1-191aa was amplified using the successfully rescued positive plasmid pACYC177-rSD17-38 of example 1 as template, rSTX2-77-191aa-F and the primer JXA1-R-NOTI-2 fu-1.
The method for connecting the amplified fragment with pACYC177-rSD17-38-F1+F2 linearization vector and the method for screening positive plasmids are the same as the construction of pACYC177-rSTX 234. The positive plasmids pACYC177-rSTX and pACYC177-rSTX2-1-191aa were obtained after construction according to the methods described above. Thus, four engineered plasmids pACYC177-rSTX234, pACYC177-rSTX, pACYC177-rSTX34, and pACYC177-rSTX2-1-191aa were obtained.
2.3 Rescue of modified Virus
BHK-21 cells were inoculated into a 12-well cell culture plate at a density of 1X 10 6 cells/well using DMEM medium containing 10% FBS in advance, and cultured in an incubator at 37℃with 5% CO 2 until the cell density reached about 80%. Cell transfection was performed as described in Lipofectamine TM 3000Transfection Reagent. 48h after cell transfection, sealing a 12-hole plate, freezing at-80 ℃, repeatedly freezing and thawing twice, taking all cell suspensions, centrifuging at 10000 Xg for 4min, and collecting supernatant. The transfection procedure was similar to that of example 1. Details are shown in Table 14.
After the BHK-21 cell freeze-thawing supernatant is obtained, the supernatant is inoculated to PAM cells to complete virus rescue. The specific method comprises the following steps: primary PAM cells were seeded at a density of 1X 10 6 cells/well in 12-well cell culture plates using 1640 medium containing 2% FBS in advance and incubated in a 5% CO2 incubator at 37 ℃. After the cells are attached, adding the freeze-thawing supernatant of the transfected BHK-21 cells, incubating for 2 hours, and discarding the liquid. 1640 medium was replaced with 2% fbs. The rescue-completed virus supernatant was obtained after 4 days.
Marc-145 cells were inoculated into 12-well cell culture plates at a density of 2X 10 5 cells/well using DMEM medium containing 10% FBS in advance, placed in an incubator at 37℃with 5% CO 2, and cultured until the cell density reached about 80%, and replaced with DMEM medium containing 2% FBS.
Mu.L of the successful rescue rSTX234,234, rSTX56,56, rSTX and rSTX2-1-191aa virus supernatant was added to Marc-145 cells and culture was continued. At the same time, rXJ and supernatant 1X 10 3TCID50 containing rSD17-38 and SD17-38 viruses successfully rescued in example 1 were inoculated with Marc-145 cells as a control group, and the cell status was observed daily. After 6 days of infection, the supernatant was collected and stored, leaving a monolayer of cells for detection of viral proteins by indirect Immunofluorescence (IFA). rXJ17-5 was detected using anti-N protein monoclonal antibody 15A1 and the engineered virus was detected using PRRSV anti-N protein monoclonal antibody 6 A1. As shown in FIG. 7, engineered viruses rSTX, rSTX successfully detected N-protein specific red fluorescence on PAM cells, but not on Marc-145 cells, demonstrating that these two engineered viruses were not suitable for Marc-145 cell culture. After rSTX234 and rSTX-1-191 aa were inoculated with Marc-145 cells, specific red fluorescence was seen for the N protein of the modified virus, whereas no fluorescence was generated for the negative control group, demonstrating that only rSTX234 and rSTX-1-191 aa modified virus acquired Marc-145 cell tropism.
Proliferation characterization of 2.4, rSTX234 and rSTX2-1-191aa on Marc-145 cells
Marc-145 cells were inoculated into 24-well cell culture plates at a density of 1X 10 5 cells/well using DMEM medium containing 10% FBS in advance, placed in an incubator at 37℃with 5% CO2 until the cell density reached about 80%, and replaced with DMEM medium containing 2% FBS.
1X 10 3TCID50 rXJ-5, rSD17-38, rSTX56, rSTX, rSTX, 234 and rSTX2-1-191aa were seeded onto Marc-145 cells in 6-well plates. 1, 2,3, 4 and 5 days after infection, 100 mu L of the supernatant is taken every day and frozen in a refrigerator at-80 ℃ for multi-step growth curve determination by a plaque method after collection. The plaque method comprises the following specific steps: marc-145 cells were inoculated into 24-well cell culture plates at a density of 8X 10 4 cells/well using DMEM medium containing 10% FBS in advance, placed in an incubator at 37℃with 5% CO2 until the cell density reached about 80%, and replaced with DMEM medium containing 2% FBS. The next day the cell density was observed until the cells reached a dense state for the next step.
100. Mu.L of virus solution at 5 time points was diluted 10-fold with 900. Mu.L of serum-free DMEM, and diluted to-5 in sequence. Then 500. Mu.L of the dilution was used to infect Marc-145 cells for 1.5h per dilution gradient. After 1.5h, the virus solution was discarded, washed gently with serum-free DMEM 1 to 2 times, 1% methylcellulose medium (30 mL medium formulation: 15mL DMEM+600. Mu.L fetal bovine serum+300. Mu.L green streptomycin+finally 15mL 2% methylcellulose) was prepared, and after shaking, the monolayer cells were directly covered. After 6 days, observation was performed. After cytopathic formation, 1% methylcellulose medium was discarded, and the medium was covered with 4% paraformaldehyde and left to stand overnight for 3 hours with crystal violet staining. After the completion of the staining, the virus particles were washed with water, observed, and counted to obtain the number of virus particles at each time point.
The results of the multi-step growth curve assay are shown in FIG. 8, with only rXJ, 17-5, rSTX, 234 and rSTX, 2-1-191aa successfully replicated on Marc-145 cells.
2.5, RSTX, 234 and rSTX2-1-191aa plaque assay
The experimental procedure for plaque is described in detail in 2.4. Plaque assay results are shown in FIG. 9, rXJ-5, rSTX234 and rSTX2-1-191aa formed plaques on Marc-145 cells, whereas rSTX, rSTX, rSD17-38 did not.
Claims (9)
1. The infectious clone plasmid pACYC177-rSD17-38 is characterized in that the infectious clone plasmid is obtained by connecting an intermediate vector with a gene segment F4, the intermediate vector is obtained by connecting gene segments F1, F2 and F3 with the plasmid pACYC177 in a homologous recombination mode, and the gene segments F1, F2, F3 and F4 are sequentially positioned at 1-1525 nucleotides, 1481-7891 nucleotides, 7844-12371 nucleotides and 12321-15177 nucleotides of the whole genome of the NADC30-LIKE PRRSV SD17-38 virus.
2. The method for constructing infectious clone plasmid pACYC177-rSD17-38 according to claim 1, characterized by comprising the following steps:
1) Respectively designing primer pairs of gene fragments F1, F2 and F3, and then respectively amplifying to obtain SD17-38-F1, SD17-38-F2 and SD17-38-F3 fragments;
2) Designing a primer pair of a gene fragment F4, firstly using a primer pair of SD17-38-Bsp1407IF4 and SD 17-38-4R 1 to amplify to obtain F4-1, then using F4-1 as a template, using a primer SD17-38-Bsp1407IF4 and a primer SD17-38-AscI-4R2 combination with 3' -end added with a gene sequence of hepatitis delta virus to amplify to obtain F4;
3) The pACYC177 plasmid is subjected to double enzyme tangential treatment by PacI and Bsp1407I, F1, F2 and F3 fragments are subjected to homologous recombination with the linearization vector to obtain plasmid pACYC177-rSD17-38-F1+F2+F3 connected with the F1, F2 and F3 fragments, then pACYC177-rSD17-38-F1+F2+F3 plasmid and F4 fragment are subjected to double enzyme digestion by Bsp1407I and AscI, and then F4 fragment and the vector are subjected to enzyme digestion connection to obtain the complete pACYC177-rSD17-38 plasmid.
3. The construction method according to claim 2, wherein the homologous recombination system comprises 20-40 ng of F1 fragment, 100-200 ng of F2 fragment, 50-150 ng of F3 fragment and 100-300 ng of pACYC177 linearization vector, and 1 mu L Exanse MultiS and 5X CE MultiS Buffuer mu L of pACYC177 linearization vector are added for 30min at 37 ℃.
4. An infectious clone virus, wherein the infectious clone is obtained by transfecting a cell with the plasmid of claim 1 and then infecting a susceptible cell.
5. The method for rescuing infectious cloned virus as claimed in claim 4, comprising the steps of: the pACYC177-rSD17-38 plasmid according to claim 1 is transfected into BHK-21 cells, and the obtained transfection solution is inoculated with primary PAM cells again, and after a few days of culture, the rescued infectious clone virus is obtained.
6. An engineered NADC30-LIKE PRRSV strain suitable for in vitro culture of Marc-145 cells, wherein the engineered strain is obtained by replacing a gene encoding part of the envelope protein of the infectious clone virus according to claim 4 with a gene encoding part of the envelope protein of the HP-PRRSV XJ17-5 isolate.
7. The modified NADC30-LIKE PRRSV strain which is suitable for in vitro culture of Marc-145 cells according to claim 6, wherein the partial coding gene of the small envelope protein after replacement comprises coding genes of amino acids 1-191 of ORF2-ORF3-ORF4 of XJ17-5 strain or ORF2 of XJ17-5 strain, the gene sequence of ORF2-ORF3-ORF4 of XJ17-5 strain is shown as nucleotide 1-1705 of SEQ ID NO.2, and the gene sequence of ORF2 of XJ17-5 strain is shown as nucleotide 1-573bp of SEQ ID NO. 2.
8. Use of the infectious clone plasmid pACYC177-rSD17-38 according to claim 1, the infectious clone virus according to claim 4, the engineered strain according to any one of claims 6 to 7 for the preparation of a vaccine for porcine reproductive and respiratory syndrome virus.
9. A vaccine against porcine reproductive and respiratory syndrome virus, comprising the infectious cloned virus of claim 4 or the engineered strain of any one of claims 6-7.
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