CN111534547A - Construction method of recombinant baculovirus expressing avian adenovirus serotype 4 spike protein F2 - Google Patents

Abstract

The invention relates to a construction method of a recombinant baculovirus expressing avian adenovirus serotype 4 spike protein F2, which comprises the following steps: (1) constructing a FAdV-4-F2 gene recombinant baculovirus expression vector; (2) packaging and passaging baculovirus expressing recombinant FAdV-4 protein; (3) transfecting a baculovirus genome carrying a FAdV-4-F2 gene into sf9 cells, culturing for 5-7 days in an incubator at 27 ℃, and observing the cytopathic condition; collecting cell lysate when the cytopathic effect reaches 70%, centrifuging to remove precipitate, and storing as seed virus; and (5) carrying out passage of the virus, and carrying out amplification culture as seed virus when the virus is transmitted to the 3 rd generation. The invention constructs a recombinant baculovirus shuttle vector containing an F2 gene by using a baculovirus expression system, transfects Sf9 cells with the recombinant baculovirus shuttle vector to obtain recombinant baculovirus rBA-F2, and identifies the expression of a recombinant protein F2 through an indirect immunofluorescence assay (IFA) and Western blot.

Description

Construction method of recombinant baculovirus expressing avian adenovirus serotype 4 spike protein F2

Technical Field

The invention relates to a construction method of a recombinant baculovirus expressing avian adenovirus serotype 4 spike protein F2, belonging to the technical field of biology.

Background

Avian Adenovirus (fowladenovirus, FAdV) belongs to the genus avian Adenovirus of the family adenoviridae, and is divided into 5 species (a-E), 12 serotypes. The FAdV-4 can be horizontally and vertically transmitted through contact, the pathogenicity to chicks is strongest, the lethality is high, and the hosts of the diseases comprise white feather broilers, broiler breeders, laying hens and yellow feather chickens. The clinical manifestations are Inclusion Body Hepatitis (IBH) and Hydropericardium Hepatitis Syndrome (HHS). With the rapid development of the poultry industry in China, the outbreak of the avian adenovirus causes serious economic loss to the poultry industry. The F2 protein is one of capsid proteins of FAdV-4, and the F2 protein has good antigenicity, is considered to be related to tissue tropism of FAdV-4 and can effectively stimulate the body to produce neutralizing antibodies. As no serological method for quickly and specifically detecting FAdV-4 and no commercial FAV-4 vaccine exist at present. In order to reduce the loss of FAdV-4 to the poultry industry in China, further research and establish a diagnosis method and produce FAdV-4 subunit vaccine and rapid diagnosis reagent, the invention establishes a foundation for establishing and producing safe and reliable FAdV-4 serological detection method and subunit vaccine suitable for popularization by constructing a recombinant baculovirus vector containing FAdV-4-F2 protein and expressing F2 protein in insect cells by using a baculovirus expression system.

Disclosure of Invention

The invention aims to provide a construction method of a recombinant baculovirus expressing avian adenovirus serotype 4 spike protein F2. The principle of the invention is that a baculovirus recombinant vector is constructed by amplifying the whole-length fragment of the avian adenovirus serotype 4 spike protein F2, and Sf9 cells are transfected to obtain the baculovirus capable of expressing the spike protein F2. The results of indirect immunofluorescence assay (IFA) and immunoblotting assay (Western-blot) showed that the recombinant baculovirus containing F2 protein amplified in Sf9 cells reacted specifically with murine monoclonal antibody against F2 protein.

The invention aims to realize the construction method of the recombinant baculovirus expressing the avian adenovirus serotype 4 spike protein F2, which is characterized by comprising the following steps:

(1) constructing a FAdV-4-F2 gene recombinant baculovirus expression vector;

designing an F2 gene amplification primer and a pFastBacHTA linearized vector amplification primer, amplifying an F2 gene amplification primer F2 gene, amplifying a pFastBacHTA linearized vector amplification primer pFastBacHTA linearized vector, connecting and transforming to escherichia coli DH5 alpha competent cells, coating the competent cells on an LB agar plate added with Amp +, performing overnight culture, identifying positive clones by colony PCR, and constructing a recombinant plasmid pFastBacHTA-FAdV-4-F2;

the F2 gene amplification primer sequence is as follows:

TTCAAAGGCCTACATGCTCCGGGCCCCTAA as upstream primer;

CTCTAGATTCTTACGGGAGGGAGGCC as downstream primer;

the pFastBacHTA linearized vector amplification primer sequence is as follows:

TAAGAATCTAGAGCCTGCAGTCTCGAG as upstream primer;

CATGTAGGCCTTTGAATTCCGCGCGCTTCG as downstream primer;

(2) packaging and passaging baculovirus expressing recombinant FAdV-4 protein;

transforming the successfully constructed pFastBacHTA-FAdV-4-F2 plasmid into a DH10Bac competent cell, and obtaining a recombinant positive clone by using a blue-white spot screening technology; selecting a single clone, carrying out overnight amplification culture at 37 ℃, extracting a recombinant baculovirus genome with F2 gene, and simultaneously taking a baculovirus transfer vector plasmid without a target fragment as a negative control; verifying the baculovirus genome by PCR amplification using pUC/M13 Forward and pUC/M13 Reverse primers;

(3) transfecting a baculovirus genome carrying a FAdV-4-F2 gene into sf9 cells, culturing for 5-7 days in an incubator at 27 ℃, and observing the cytopathic condition; collecting cell lysate when the cytopathic effect reaches 70%, centrifuging to remove precipitate, and storing as seed virus; and (5) carrying out passage of the virus, and carrying out amplification culture as seed virus when the virus is transmitted to the 3 rd generation.

Through the invention, the purpose of the invention is realized through the following technical scheme:

(1) primers were designed to amplify the serum type 4 avian adenovirus F2 gene and pFastBacHTA linearized vectors, the specific primer sequences are shown in Table 1 and synthesized by Suzhou Jinzhi Biotech Co.

(2) Cloning the F2 gene to a pFastBacHTA vector by a one-step cloning method;

(3) transforming the plasmid obtained in the step into a DH10Bac competent cell to prepare a recombinant shuttle plasmid (rBa-F2);

(4) rBa-F2 is transfected into insect cells to obtain recombinant baculovirus expressing serum 4 type adenovirus F2 gene and express recombinant protein.

(5) And identifying immunoreactivity of the recombinant protein expressed by the virus by a Western-blot and IFA method.

The avian adenovirus serotype 4 fiber protein F2 is expressed by the baculovirus vector, has high expression level, contains a His label, can facilitate the later purification step, can be prepared into avian adenovirus type 4 subunit vaccines, and can also be used as an envelope antigen in the preparation and development of FAdV-4 enzyme-linked immunosorbent reagents.

Description of the inventive principles:

the baculovirus expression system is prepared by infecting Sf9 cells with repackaged baculovirus carrying exogenous gene F2 by using an in vitro recombination technology, so that the recombinant baculovirus is amplified in Sf9 cells, is released to the outside of the cells and exists in cell supernatant, and foreign proteins can be purified by means of His tag proteins at the later stage. The foreign protein expressed by the system has natural conformation, high-purity and high-concentration protein can be obtained by protein purification, and the purified product is beneficial to muscle or oral immunity.

Therefore, the invention selects F2 as an expression object to obtain F2 protein in a natural conformation, and the F2 protein is used as a candidate vaccine for preventing FAdV-4 infection and an antigen substance for detecting antibodies of FAdV-4.

Compared with the prior art, the invention has the following beneficial technical effects:

(1) based on the advantages that an insect baculovirus expression system is superior to a prokaryotic expression system, the protein has complete post-translational processing modification capacity when expressing exogenous protein, expresses FAdV-4-F2 protein on insect cells, has natural conformation, and has better immunogen effect of simulating FAdV-4;

(2) the recombinant protein prepared by the baculovirus expression system has high expression level in Sf9 insect cells and is easy to purify.

The invention constructs a recombinant baculovirus shuttle vector containing an F2 gene by using a baculovirus expression system, transfects Sf9 cells with the recombinant baculovirus shuttle vector to obtain recombinant baculovirus rBA-F2, and identifies the expression of a recombinant protein F2 through an indirect immunofluorescence assay (IFA) and Western blot. The invention provides a construction method of recombinant baculovirus expressing serum 4 type avian adenovirus spike protein F2, which has high protein expression amount in Sf9 insect cells and easy purification, and the expressed protein has natural conformation and similar antigenicity to natural virus antigenicity, can provide candidate antigens for further developing serum 4 type avian adenovirus genetic engineering subunit vaccines, and simultaneously provides basic test materials for further researching FAdV-4 pathogenesis.

Drawings

FIG. 1 is an amplification diagram of the F2 gene; wherein: m is Super DNA Marker; 1 is the F2 amplified fragment.

FIG. 2 shows primers M13F and M13R for detecting recombination of a target gene in baculovirus; wherein: m: super DNAmarker; lane 1, rBacmid-FAdV-4-F2; lane 2, rBacmid-HTA control; lane 3, rBacmid-DNAControl.

FIG. 3 is a Western blot expression identification chart of recombinant proteins; wherein: m is Protein Marker; lane 1 Sf9 cell lysate infected with rBacmid-FAdV-4-F2 recombinant baculovirus; lane 2, rBacmid-HTA control; lane 3 Sf9 control.

FIG. 4 is an indirect immunofluorescence assay of recombinant baculovirus rBacmid-FAdV-4-F2 infected Sf9 cells.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings:

1 Material

1.1 strains, plasmids, cells

The avian adenovirus serotype 4 JH is isolated and identified in the laboratory. DH5 alpha competent cells, DH10Bac competent cells, Sf9 insect cells and pFastBacHTA plasmids are all preserved in the laboratory

1.2 Primary reagents

Monoclonal antibodies against the F2 protein were stored in the laboratory; phanta Super-Fidelity DNA polymerase, Gene1Kb Super Marker, 2 XTAQA Master Mix, and ExnaseTMII ligase were purchased from Nanjing Novowed King; the plasmid mini-extraction kit is purchased from AxyPrep company; gel recovery DNA kit was purchased from QIAGEN; lipofectamine TM3000 transfection reagent, FBS fetal bovine serum purchased from Gibco company; goat anti-mouse FITC labeled IgG was purchased from SIGMA; western-blot exposure solution was purchased from Nanjing Philid Biotech; gentamicin (GEN), kanamycin (Kan), tetracycline (TET), X-gal were purchased from Shanghai Biotech.

1.3 Main Medium

The LB liquid culture medium, the LB liquid culture medium containing Amp, the LB solid culture medium containing Amp, the SOC culture medium, the selective LB liquid culture medium and the selective LB solid culture medium are prepared by the laboratory. SF900 medium was purchased from Gibco.

1.4 design and Synthesis of primers

Primers for amplifying pFastBacHTA linearized vector and FAdV-4 avian adenovirus F2 protein gene fragment are designed according to FAdV-4 sequence accession number base sequence, and specific primer sequences are shown in Table 1

TABLE 1 PCR amplification linearized pFast-Bac-HTA and avian adenovirus F2 gene primers

2. Method of producing a composite material

2.1 amplification of the Gene of interest

Firstly, preparing an avian adenovirus genome, splitting a virus of a separated strain of avian adenovirus serotype 4 by using a cell lysate, then extracting the virus genome by using phenol, chloroform and isoamylol, finally precipitating by using absolute ethyl alcohol, dissolving in 30uL sterilized ultrapure water to obtain the avian adenovirus genome, and placing at the temperature of minus 20 ℃ for later use. mu.L of extracted DNA of avian adenovirus serotype 4 was used as a template for PCR amplification of the spike protein gene F2 using a 50. mu.L PCR system. The reaction conditions are as follows: DNA template 1. mu.L, 5 XBuffer 10. mu.L, dNTP Mix 1. mu.L, primers 2. mu.L each, high fidelity enzyme 1. mu.L, sterile ultrapure water was added to 50. mu.L. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 4min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 3min for 30 cycles; extension at 72 ℃ for 10 min. Meanwhile, pFastBacHTA vector plasmid is used as a template, and a linearized pFastBacHTA vector is amplified by PCR. The PCR reaction system, conditions and procedures were identical to those of the PCR amplification of F2 gene. Carrying out electrophoresis on the PCR product by 1% agarose gel, wherein the voltage is 90V, and the electrophoresis time is 45 min; as shown in FIG. 1, a single band appeared at 1440bp, confirming that the desired gene fragment of interest was obtained.

2.2 construction of cloning vector and identification of recombinant donor plasmid pFastBacHTA-FAdV-4-F2

After the target fragment obtained by amplification in the step 2.1 and pFastBacHTA are linearized and connected at room temperature for half an hour, the connection product is transformed into DH5 alpha competent cells and then coated on a plate, the plate is inverted and cultured overnight in a constant-temperature incubator at 37 ℃, a single colony is selected for PCR identification, a cloning vector pFastBacHTA-FAdV-4-F2 is obtained when PCR is positive, and a bacterial liquid with PCR positive is extracted by adopting a small upgrading particle extraction step of AxyGEN company to extract positive plasmids for storage. The sequencing and the identification of the recombinant plasmid prove that the recombinant donor plasmid pFastBacHTA-FAdV-4-F2 is successfully constructed.

2.3 construction and identification of recombinant baculovirus shuttle plasmid rBacmid-FAdV-4-F2

The recombinant donor plasmid is transformed into DH10Bac competent cells in SOC culture medium, shaking culture is carried out for 4h at 37 ℃ and 225rpm, the culture is sequentially diluted by the SOC culture medium for three concentration gradients, 200 mu L of the culture is respectively and evenly coated on LA-Bac plates containing kanamycin (50 mu g/mL), tetracycline (10 mu g/mL), gentamicin (7 mu g/mL), IPTG (100mM) and Bluo-gal, and culture is carried out for 72h at 37 ℃ until bacterial colonies are completely appeared. Screening blue white spots, selecting positive white clones, purifying on a fresh LA-Bac selective solid culture medium, and culturing at 37 ℃ for 48-72 h; and continuously purifying for 3 generations until no blue colony appears, picking up a single colony, and performing colony PCR identification to obtain positive bacteria with correct identification. The recombinant plasmid of the positive bacteria extracted by the alkaline lysis method is subjected to PCR identification by using M13F/M13R, the result is shown in figure 2, the amplification product is 4440bp, the successful transposition of the target gene is proved, and the construction of the recombinant plasmid rBacmid-FAdV-4-F2 is successful.

2.4 expression of recombinant plasmids in insect cells

Transfecting Sf9 insect cells by the recombinant rod-shaped plasmid, gently blowing and stirring SF900 culture medium, plasmid DNA and Lipofectamine TM3000 uniformly, and standing for 15min at room temperature; adding the mixture to a monolayer of 80% -90% cells in a 6-well plate; culturing in 27 deg.C incubator for 7 d; collecting culture supernatant, namely P1 generation recombinant baculovirus rBacmid-FAdV-4-F2, transmitting the strain to the 3 rd generation, collecting culture supernatant and cell sediment, carrying out Western-Blot analysis on the cell sediment to obtain an expression product, and subpackaging corresponding cell supernatant containing the recombinant baculovirus at-80 ℃ for seed virus when a specific band appears.

2.5 identification of expression products

1) Western-blot identification

Centrifuging the cells at 10000rpm/min for 5min, respectively collecting cell precipitate and supernatant, adding 100 μ L of cell lysate into the cell precipitate, mixing, immediately performing ice bath lysis, adding 6 × Buffer 20 μ L after 30min, mixing, and decocting in a constant temperature metal bath at 98 deg.C for 10min until no viscosity exists; the samples were then separated by SDS-PAGE and Western-Blot analysis was performed on proteins transferred to NC nitrocellulose using a transprinter. Sealing the protein-transferred NC membrane with 5% skimmed milk at room temperature for 2h, diluting with 5% skimmed milk to 1:1500, incubating at 4 deg.C overnight, washing the membrane with PBST for 3 times 5 min/time the next day; after washing, incubating a secondary antibody, wherein the secondary antibody is a commercial goat anti-mouse HRP-labeled secondary antibody, diluting the secondary antibody to 1:10000 by using 5% skim milk, incubating for 1h at normal temperature, washing the membrane by using PBST (para-phenylene benzobisoxazole) for 3 times and 5 min/time; and finally, taking a picture in a chemiluminescence imaging system after the ECL luminescent liquid is developed. As shown in FIG. 3, the F2-specific protein band appeared at a protein molecular weight of about 70kDa in Sf9 cell lysate transfected with rBacmid-FAdV-4-F1, whereas no specific band appeared in control cells transfected with pFAST-BacHTA vector and normal Sf9 cells.

2) Indirect immunofluorescence assay (IFA)

Cells transfected with passage 3 recombinant virus and maintained in culture for 2 days were plated in 48-well plates with pre-chilled acetone: fixing with ethanol (3: 2) fixing solution, drying, diluting with anti-F2 protein mouse monoclonal antibody at a dilution of 1:1000, 200 μ L per well, and acting at 37 deg.C for 45 min; the primary antibody was discarded, washed 3 times with PBS, added with 200. mu.L of goat anti-mouse FITC-labeled secondary antibody diluted 1:200, allowed to act at 37 ℃ for 45min, discarded, washed 3 times with PBS, observed on an inverted fluorescence microscope and photographed. As shown in FIG. 4, specific bright green fluorescence was observed in both Sf9 cells transfected with recombinant rBacmid-FAdV-4-F2, and not in the control cells and normal Sf9 cells transfected with wild-type baculovirus of rBacmid-HTA.

Western blot and IFA both prove that the recombinant baculovirus can successfully express the target protein F2.

Sequence listing

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Claims (1)

1. A construction method of recombinant baculovirus expressing avian adenovirus serotype 4 spike protein F2 is characterized by comprising the following steps:

(1) constructing a FAdV-4-F2 gene recombinant baculovirus expression vector;

designing an F2 gene amplification primer and a pFastBacHTA linearized vector amplification primer, amplifying an F2 gene amplification primer F2 gene, amplifying a pFastBacHTA linearized vector amplification primer pFastBacHTA linearized vector, connecting and transforming to escherichia coli DH5 alpha competent cells, coating the competent cells on an LB agar plate added with Amp +, performing overnight culture, identifying positive clones by colony PCR, and constructing a recombinant plasmid pFastBacHTA-FAdV-4-F2;

the F2 gene amplification primer sequence is as follows:

TTCAAAGGCCTACATGCTCCGGGCCCCTAA as upstream primer;

CTCTAGATTCTTACGGGAGGGAGGCC as downstream primer;

the pFastBacHTA linearized vector amplification primer sequence is as follows:

TAAGAATCTAGAGCCTGCAGTCTCGAG as upstream primer;

CATGTAGGCCTTTGAATTCCGCGCGCTTCG as downstream primer;

(2) packaging and passaging baculovirus expressing recombinant FAdV-4 protein;

transforming the successfully constructed pFastBacHTA-FAdV-4-F2 plasmid into a DH10Bac competent cell, and obtaining a recombinant positive clone by using a blue-white spot screening technology; selecting a single clone, carrying out overnight amplification culture at 37 ℃, extracting a recombinant baculovirus genome with F2 gene, and simultaneously taking a baculovirus transfer vector plasmid without a target fragment as a negative control; verifying the baculovirus genome by PCR amplification using pUC/M13 Forward and pUC/M13 Reverse primers;

(3) transfecting a baculovirus genome carrying a FAdV-4-F2 gene into sf9 cells, culturing for 5-7 days in an incubator at 27 ℃, and observing the cytopathic condition; collecting cell lysate when the cytopathic effect reaches 70%, centrifuging to remove precipitate, and storing as seed virus; and (5) carrying out passage of the virus, and carrying out amplification culture as seed virus when the virus is transmitted to the 3 rd generation.

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