CN110184287B - Method for preparing recombinant virus and application thereof - Google Patents
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- CN110184287B CN110184287B CN201910443328.3A CN201910443328A CN110184287B CN 110184287 B CN110184287 B CN 110184287B CN 201910443328 A CN201910443328 A CN 201910443328A CN 110184287 B CN110184287 B CN 110184287B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a method for preparing recombinant virus, which takes a virus vector as a matrix, firstly introduces bidirectional screening genes through Red/ET recombination, further recombines and replaces the bidirectional screening genes with target genes to obtain recombinant plasmids containing target genes and virus genome, and further saves and obtains the recombinant virus. The method has the advantages that the method is simple in steps, can accurately and rapidly realize construction of recombinant viruses, can accommodate exogenous genes, realizes multi-site directional insertion, has no trace insertion and convenient screening marking, and finally forms the recombinant viruses capable of expressing multiple antigen genes, and is applied to carrier vaccines.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and in particular relates to a method for preparing recombinant viruses and application thereof.
Background
Vaccination is one of the important ways to isolate disease transmission channels between animals and humans. At present, various vaccines, including genetically engineered vaccines, have been developed for different epidemic disease types in China. Along with the rapid development of recombinant technology, genetic engineering vaccines are also widely applied to the prevention and treatment of animal infectious diseases, and a solid foundation is laid for improving the effect of preventing and treating infectious diseases.
In recent years, recombinant virus vector vaccines are widely paid attention to, and the recombinant virus vector vaccines are obtained by inserting exogenous protective antigen genes into viral genomes, and express corresponding target proteins after the recombinant viruses immunize organisms, so that immune response is induced. The recombinant virus vector vaccine has the advantages of long insertion foreign gene, multiple inoculation paths, capability of inducing humoral immunity and cellular immune response, easiness in production and preparation and the like, can overcome certain defects of the traditional vaccine, can greatly improve the disease preventive property, and has been widely applied to vaccine development.
The main modification techniques currently used in viral genetic engineering have the following four aspects:
disadvantages of RecA protein-mediated homologous recombination are: firstly, a longer homologous arm is needed, the operation is limited only by using escherichia coli, the operation steps are complex and complicated, the false positive rate is high, and the like; cre/loxp mediated modification technique: the method has the advantages of space-time specificity, high efficiency (not influenced by the size and position of a target gene), accuracy (less nonspecific recombination) and rapidness, and has the defects of complicated steps and high cost; the Tn transposon can mediate gene mutation and random insertion, and has the defects of incapability of fixed-point insertion, difficulty in controlling the copy number of insertion and low efficiency. It can be seen that common homologous recombination requires homology arms of up to several hundred and even several thousand bases, and is subject to restriction by restriction sites, and is not flexible to use.
Disclosure of Invention
The present invention is directed to the above problems, and provides a method for preparing a recombinant virus, which is rapid, accurate and simple to operate, and is characterized in that the method comprises the following steps:
s1: carrying out homologous recombination on the genome of the virus to be selected and a receptor vector to obtain a virus vector plasmid;
s2: selecting a plasmid containing a bidirectional selection gene, and designing a primer for the bidirectional selection gene to amplify to obtain an amplification product of the bidirectional selection gene containing a homology arm, wherein the bidirectional selection gene comprises a resistance selection gene and a reverse selection gene, the resistance selection gene comprises one or more of kanamycin resistance gene (Knr), benzyl mycin resistance gene (Amp), hygromycin (Hyg) or chloramphenicol resistance gene (Cmr), the plasmid containing the bidirectional selection gene is different from the resistance of the receptor vector in the step S1, and the reverse selection gene comprises one or more of CcdB, scaB, rpl, tetr, phes, galk, thyA or TolC;
s3: transferring the virus vector plasmid in the step S1 and the amplification product in the step S2 into competent cells 1 together, and performing resistance screening to obtain recombinant plasmids containing bidirectional screening genes and virus vector genes, wherein the competent cells 1 are engineering bacteria which can accommodate the reverse screening genes in the step S2 and can express Red alpha/beta recombinase;
s4: and (3) transferring the gene expression cassette containing the homologous arm target gene and the recombinant plasmid in the step (S3) into competent cells (2), screening to obtain recombinant plasmids with HA replaced by the bidirectional screening genes, and saving to obtain recombinant viruses which are simultaneously immunized with the virus to be selected and loaded with antigens, wherein the competent cells (2) are engineering bacteria capable of expressing Red alpha/beta recombinase but incapable of accommodating the reverse screening genes in the step (S2).
The method is to obtain recombinant virus containing target genes by two-step homologous recombination by using RED/ET recombination technology and ccdB reverse screening technology on the basis of an infectious cloned virus vector. The recombination system is safe and reliable, and can realize stable expression.
Among the above methods, the method of homologous recombination between the genome of the virus to be selected in step S1 and the receptor vector is known, and the conventional homologous recombination is carried out in an engineering bacterium containing a recombinase by introducing homology arms homologous to the target gene at both ends of the insertion site of the receptor vector.
Further, the genome in the step S1 is DNA or cDNA.
The method is suitable for viruses with DNA as main genetic material and viruses with RNA as main genetic material, and the steps can be carried out only by reverse transcription of RNA into cDNA.
Further, the receptor vector in the step S1 is one of a plasmid vector, a phage vector or an artificial chromosome vector.
Further, the bidirectional screening gene in the step S2 is ccdB-amp, and the competent cell 1 in the step S3 is GBred gyrA462 E.coli.
The Ccd operon exists on F plasmid of escherichia coli, is a toxin-antitoxin system, and consists of ccdA and ccdB, wherein ccdB is toxic protein, only expression of ccdB gene can cause death of general host cells (such as competent cells 2), and recombinant plasmid containing the ccdB gene and plasmid replaced by ccdB gene are selected through different accommodations of the ccdB gene to the ccdB gene (such as the ccdB gene can be expressed and survived in competent cells 1), so that the screening step is simplified, the positive rate is high, and the use is simpler and more efficient.
Further, in the step S3, homology arms are positioned at two ends of the insertion site of the virus to be selected, and the nucleotide sequence of the homology arms is 35-50 bp.
Use of said method for preparing recombinant viruses in the preparation of vaccines.
When the method for preparing recombinant viruses is applied to the field of vaccines, the virus to be selected in the step S1 is safe to the immunized host, can cause effective immune response and can contain exogenous genes, such as HVT, IBV, NDV, PRV or ADV.
Further, the vaccine is an infectious bronchitis virus expressing the HA gene of the H9N2 avian influenza virus.
The sequence of the HA gene is shown as SEQ ID No.1, and the HA gene is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: v201931.
Further, the rH120-delta 5a/H9HA is prepared by the following method:
s1: constructing a viral plasmid vector p15A-H120 of an infectious bronchitis virus vaccine strain H120 by utilizing a plasmid vector p 15A;
s2: amplifying an amp-ccdB bidirectional screening marker gene expression cassette containing a homology arm by taking a plasmid pBR322-kanR-amp-ccdB-rpsLneo as a template;
s3: transferring the viral plasmid vector p15A-H120 in the step S1 and the amp-ccdB bidirectional screening marker gene expression cassette in the step S2 into competent GBred gyrA462 somatic cells together, and carrying out homologous recombination to obtain a recombinant plasmid p15A-H120-amp-ccdB;
s4: transferring a gene expression cassette containing an HA gene shown as SEQ ID No.1 and the plasmid p15A-H120-amp-ccdB obtained in the step S3 into competent GBdir E.coli together, and screening to obtain recombinant plasmid p15A-H120-HA;
s5: constructing helper plasmid pVAX1-H120N, transfecting BSR-T7 cells with the recombinant plasmid p15A-H120-HA and the helper plasmid pVAX1-H120N in the step S4, collecting the transfected supernatant, transferring the supernatant into chick embryos, and carrying out passage to obtain the infectious bronchitis virus rH 120-delta 5A/H9HA for expressing the HA gene.
In order to achieve the above purpose, the present invention specifically adopts the following technical scheme:
firstly constructing an infectious cloning vector containing H120 whole genome cDNA, firstly, in GBred gyrA462 escherichia coli expressing recombinant protein Red alpha/Red beta, replacing a 5A gene in p15A-H120 by a screening marker gene amp-ccdB through line-loop recombination to obtain p 15A-H120-delta 5A/amp-ccdB, and preparing a linearization vector p 15A-H120-delta 5A/amp-ccdB by enzyme digestion. And secondly, in GBred escherichia coli, the target gene and a linear vector p 15A-H120-delta 5A/amp-ccdB are recombined by utilizing line-loop recombination, so that the amp-ccdB gene is replaced by an exogenous gene, and p 15A-H120-delta Sa/H9HA with an insertion site at 5A is obtained. Thirdly, the recombinant virus rH120-delta 5a/H9HA is obtained through rescue.
Compared with the prior art, the invention has the following advantages and effects:
the method has simple steps, can accurately and rapidly realize the construction of the recombinant virus, and the successfully constructed recombinant virus has the advantages of accommodating a plurality of large fragment exogenous genes, realizing multi-site directional insertion, no trace insertion and convenient screening and marking, and finally forms the recombinant virus capable of expressing a plurality of antigen genes, and is applied to carrier vaccines.
Biological material preservation information
Classification naming: infectious bronchitis Virus IBV rH120-Delta5a/H9 HA, latin brand name: infectious bronchitis virus, accession number: china center for type culture collection, preservation address: university of martial arts, preservation date: and the preservation number is CCTCC NO: v201931.
Drawings
FIG. 1 shows the preparation of a selectable marker gene containing homology arms by PCR amplification, wherein 1: Δ5a/amp-ccdB;2: blank control; m: DNA Marker DL2,000.
FIG. 2 is the identification of recombinant plasmid p15A-H120- Δ5a/amp-ccdB, wherein A: p 15A-H120-delta 5A/amp-ccdB restriction SnapGene software prediction, B: cleavage of p15A-H120- Δ5a/amp-ccdB by XhoI and Bstz 17I; m: NEB 1Kb DNA Ladder.
FIG. 3 is a functional verification of the reverse screening marker gene.
Fig. 4: preparation of H9HA gene containing homology arms for PCR amplification, 1: Δ5a/H9HA; 2: blank control; m: DNA Marker DL2,000.
FIG. 5 is a recombinant plasmid p15A-H120- Δ5a/H9HA restriction enzyme assay wherein A: p 15A-H120-delta 5A/H9HA cleavage SnapGene software prediction, B: cleavage of p 15A-H120-delta 5A/H9HA by XhoI and Bstz 17I; m: NEB 1Kb DNA Ladder.
FIG. 6 shows PCR amplification of rH 120F 5 strain and H120-. DELTA.5a/H9 HA F5 strain S1, M and 5ab genes, wherein 1-3: the rH 120F 5 strain S1, M and 5ab genes; 4-6: h120-delta 5a/H9HA F5 strain S1, M and delta 5a/H9HA gene; 7: a negative control; m: DNA Marker DL5,000.
FIG. 7 is an indirect immunofluorescence assay for exogenous gene expression of rH120- Δ5a/H9HA F5 recombinant virus wherein A: CK cells infected with recombinant virus rH120-delta 5a/H9HA F5; b: CK cells infected with rH 120F 5; c: blank CEF cells
FIG. 8 shows proliferation curves of recombinant virus rH120- Δ5a/H9HA F5 chick embryos.
FIG. 9 is a schematic diagram of the pBR322-kanR-amp-ccdB-rpsLneo structure.
Fig. 10 is a schematic diagram of p15A structure.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
The reagents and materials used in the present invention are commercially available unless otherwise specified.
Example 1
1) Construction of infectious clone p15A-H120
Three complete gene sequences (comprising 5'-UTR, 3' -UTR and Ploy (a)) were published according to Genbank: FJ888351, FJ807652 and GU393335, the H120 complete gene sequence is divided into 4 segments and cloned into pBR322 vector containing homology arms to form pBR 322-H120A-D, and the overlapping region of 50-70bβ is contained between two adjacent segments as recombination homology arms. The first 5' -end H120-A was added with the T7 promoter by PCR during cloning. Each fragment clone needs to be subjected to enzyme digestion identification and sequencing to verify the sequence correctness, and then four fragment DNA (H120-A-D) is obtained through enzyme digestion. The sequence of HDVR-T7 ter containing the genome 3' homology arm was amplified as the 5 th sequence of the H120 infectious clone by PCR using the plasmid pUC57-HDVR-T7 ter as a template. H120-A-D, HDVR-T7 ter and a linear vector p15A containing a homology arm are assembled in E.coli by utilizing RED/ET recombination technology, so as to form an infectious clone p15A-cm-T7 precursor-H120 genome-HDVribozyme-T7 terminator (p 15A-H120 for short).
2) PCR amplification of the selectable marker Gene Δ5a/amp-ccdB
The screening marker gene delta 5a/amp-ccdB containing homology arms was amplified by PCR using the plasmid pBR322-kanR-amp-ccdB-rpsLneo structure as a template, as shown in FIG. 9. The primers used were as follows:
Δ5a/amp-ccdB-F：
5‘ACCTACACTACTTACTTGTAATAAGGGCGTTTGGACTTACAAGCGCTTAACAAATACAGACGGCGATCGCTTTGTTTATTTTTCTAAATAC3’
Δ5a/amp-ccdB-R：
5‘CTGGCTTTTTTTTGAACAAAGCGATCGCGCATATACGCCCACCCAATCGCTGGTATGAATAATAGTAAAGATAATCCTTTTCGCGGAGCAAT’3
the reaction system is shown in Table 1, and the PCR amplification parameters are 98 ℃ for 2min;98 ℃ for 10s;55 ℃ for 5s;72 ℃,20s; amplifying for 35 cycles; finally, the mixture was extended at 72℃for 5min. The PCR product was taken 5uL and the amplification result was observed by 1% agarose gel electrophoresis, the result is shown in FIG. 1, the remaining PCR product was recovered and purified by using a PCR product recovery kit, the nucleic acid concentration was measured, and the sequence correctness was verified by sequencing.
TABLE 1 PrimeSTAR Max DNA Polymerase polymerase PCR reaction System
The electrophoresis result shows that the PCR obtains the screening marker gene ccdB-amp containing the homology arm. The PCR products were electrophoresed on a 1% agarose gel, with a size of about 1.5Kb, consistent with the expectations (FIG. 1).
Recovery of purified PCR products with restriction enzyme DpnI digestion to remove plasmid templates, enzyme cutting system according to Table 2, 37 ℃ reaction for 2 hours, enzyme cutting products through 1% agarose gel electrophoresis observation of amplification results, and gel recovery kit recovery and purification of enzyme cutting products, determination of nucleic acid concentration.
TABLE 2 digestion identification
3) Screening marker gene and p15A-H120 electric conversion GBred gyrA462 competence
GBred gyrA462 electrotransformation competent preparation: a sterile 2mL centrifuge tube was taken, the lid was perforated with a 2mL syringe needle, 1.8mL of LB medium containing streptomycin was added, GBred gyrA462 single colonies were picked up and inoculated into the 2mL centrifuge tube, and incubated overnight at 30℃with shaking at 260 rpm. The next day, taking a sterile 1.5mL centrifuge tube, punching a cover by using a 2mL syringe needle, adding 1.4mL LB culture medium without antibiotics, adding 40 mu L of overnight bacterial liquid, shaking at 30 ℃ and 260rpm for 2 hours, adding 35 mu L of 10% L-arabinose solution, shaking at 37 ℃ and 260rpm for 50 minutes, and inducing to express the Red alpha/Red beta recombinant protein. After that, the bacterial liquid is put on ice, sterilized pure water is pre-cooled in advance, a centrifuge is pre-cooled to 2 ℃ in advance, and the bacterial liquid is centrifuged at 2 ℃ at 9000rpm for 30s; discarding the supernatant, adding 1ml of pre-chilled pure water, blowing to resuspend the thalli, centrifuging the thalli for the second time at 2 ℃, and performing 10000rpm for 30s; the supernatant was discarded, 1ml of pre-chilled water was added, the resuspended cells were blown off, the cells were centrifuged a third time at 2℃at 11000rpm for 30s, the supernatant was discarded, and 30. Mu.L of pre-chilled water was added for resuspension. The competent preparation should be placed on ice and used immediately.
The first recombination process: placing the clean and sterile electric shock cup in an ultra clean bench, ventilating and drying, and pre-cooling on ice. Screening marker genes ccdB-amp and p15A-H120 are added into freshly prepared GBred gyrA462 electric transferring competence by dialing 1.5mL centrifuge tube bottom by hand, gently mixing, transferring into a 1mm electric shock cup, placing the electric shock cup into an electric transferring instrument, and carrying out electric transferring according to a set RED/ET electric transferring program, wherein electric transferring parameters are 1350V,10 mu F and 600 Ohms. Immediately after electrotransfer, 1mL of SOC medium was added to the cuvette and the bacteria resuspended and transferred to a 2mL centrifuge tube. Placing the bacterial liquid after electric transformation on a shaking table at 37 ℃, and shaking and culturing at 260rpm for 60min to finish recombination and resistance recovery. Placing the bacterial liquid in a centrifuge, centrifuging at 3000rpm for 1min to precipitate bacterial cells, absorbing most of supernatant, retaining 200 mu L of supernatant, blowing and resuspension, completely coating on an LB plate containing ampicillin, placing the plate in 37 ℃ for culture, and performing normal culture for 1h and then performing inversion culture for 12-16h.
Screening recombinant plasmids containing screening marker genes: colonies were picked up from the plates, inoculated into 500. Mu.L of an LB liquid medium containing ampicillin, cultured at 37℃under shaking at 260rpm for 6 hours, and 100. Mu.L of the bacterial liquid was inoculated into 10mL of an LB liquid medium containing ampicillin for expansion culture. After shaking culture for 6 hours at 37 ℃, using a plasmid miniprep kit to extract plasmids from 5mL of bacterial liquid, carrying out restriction enzyme digestion identification, preparing an enzyme digestion system according to Table 3, reacting for 2 hours at 37 ℃, observing an amplification result of enzyme digestion products through 1% agarose gel electrophoresis, identifying correct plasmids, sending the plasmids to a large gene company for sequencing, and naming the recombinant plasmids with correct sequencing as p 15A-H120-delta 5A/amp-ccdB.
TABLE 3 enzyme digestion identification system
The result shows that the linear selection marker gene ccdB-Amp and the circular p15A-H120 plasmid undergo linear-circular recombination in GBred gyrA462 escherichia coli expressing Red alpha/Red beta recombinant protein, the selection marker gene is replaced with the 5A gene to generate a recombinant plasmid, and the correct recombinant plasmid is selected through Amp resistance selection, plasmid enzyme digestion identification (figure 2) and sequencing identification. The recombinant plasmids were designated as p15A-H120-ccdB-amp, respectively.
And (3) functional verification of screening marker genes: the prepared recombinant plasmid p15A-H120-ccdB-amp is respectively electrically transformed into GBred gyrA462 and GBred electrotransformation competence (without inducing expression of recombinant protein). After recovery from transformation, the bacterial solution was spread on LB plates containing ampicillin, and the plates were incubated at 37℃overnight to observe E.coli growth.
Recombinant plasmid p 15A-H120-delta 5A/amp-ccdB was transformed electrically into GBred and GBred gyrA462 for competent recovery, and the GBred gyrA462 was cultured overnight with plating, so that a large number of colonies could be grown, but GBred could not or very little colonies could be grown (FIG. 3), indicating that the reverse screening marker gene ccdB could be expressed normally.
The GBred gyrA462 of electrotransformation p 15A-H120-delta 5A/amp-ccdB grows normally, a large number of colonies can grow, and GBred cannot grow or only grows a very small number of colonies.
4) Recombination of antigen gene replacement screening gene
Amplification of antigen genes containing homology arms Δ5a/H9 HA:
RNA of avian influenza A/chicken/Guangdong/YF/2018 was extracted according to the instructions of RNeasy plus Mini Kit.
The RNA is subjected to reverse transcription according to the specification of a reverse transcription kit to obtain cDNA as a template, and the HA gene containing a homology arm is obtained through amplification.
Primer: Δ5a/H9 HA-F:
ACCACCTACACTACTTACTTGTAATAAGGGCGTTTGGACTTACAAGCGCTTAACAAATACAGACGATGGAGACAGTATCACTAATAAC
Δ5a/H9 HA-R：
CTCTAATCCTTCTCTCAGATAAATTCGCGCTTTTCTTGCTATTGCTCCGCGAAAAGGTTATTATATACAAATGTTGCATCTG
the reaction system is as in table 1: the PCR reaction conditions are that the pre-denaturation is carried out at 98 ℃ for 5min, the denaturation is carried out at 98 ℃ for 10s, the annealing is carried out at 55 ℃ for 5s, and the extension is carried out at 72 ℃ for 20s, so that 35 cycles are completed; extending at 72℃for 10min.
The amplification results are shown in FIG. 4.
GBred electrotransformation competent preparation: the preparation process is similar to GBred gyrA462 electrotransformation competent preparation, and L-arabinose is also required to induce and express Red alpha/beta recombinant protein. The competent preparation should be placed on ice and used immediately.
The second recombination process: placing the clean and sterile electric shock cup in an ultra clean bench, ventilating and drying, and pre-cooling on ice. HA genes delta 5A/H9HA containing homology arms and linear vectors p 15A-H120-delta 5A/amp-ccdB are added into GBred electrotransformation competence which is just prepared by 500ng respectively, and electrotransformation and resuscitation are carried out according to a first recombination method and program. The resuscitated bacterial solution is centrifuged at 3000rpm for 1min to precipitate the bacterial cells, most of the supernatant is sucked and removed, 200 mu L of supernatant is reserved and is blown to be resuspended, the whole is coated on an LB plate containing chloramphenicol, and the plate is placed at 37 ℃ for culture, and is firstly placed vertically for 1h and then is cultured reversely for 12-16h.
Recombinant plasmid selection
And (5) picking a colony from the flat plate, expanding and shaking bacterial liquid, extracting plasmids, and carrying out enzyme digestion identification. The results of the enzyme digestion are shown in FIG. 5, 5mL of the identified correct bacterial liquid is added into 200mL of LB liquid medium containing chloramphenicol for expansion culture, and after shaking culture for 16h at 37 ℃ and 260rpm, plasmids are extracted according to the instruction of a large amount of endotoxin-free plasmid extraction kit and are frozen at-20 ℃. And taking a small amount of plasmid to send to large gene company for sequencing and verifying the recombinant gene part. The correct recombinant plasmid was verified to be designated p15A-H120- Δ5a/H9HA.
5) Rescue of recombinant viruses
BSR-T7 cells were passaged to six well plates one day prior to transfection, and when cells grew to around 80%, the medium was changed to 2% serum, GMEM medium without diabodies and G418. 1ug each of recombinant plasmid p15A-H120-HA and helper plasmid pVAX1-H120N (DAN transfection amount per well of six-well plate) was taken, a transfection system was prepared according to Lipofectamine 3000 Reagent (invitrogen) instructions and added to a cell culture solution, and after cells were placed at 37℃in a 5% CO2 incubator for 4 hours, the medium was changed to 2% serum, and the culture was continued in a G418-containing GMEM medium. After 48h, the culture dish is frozen at-80 ℃, thawed at normal temperature, the cells are broken twice by repeated freezing and thawing, and the cell supernatant is obtained and named as F0. The cell supernatant was inoculated with 5 SPF chick embryos of 10 days old, 0.2 ml/chicken embryo, and incubated at 37 ℃. Chick embryos were observed 24 hours after inoculation and dead chick embryos were discarded. The chick embryo allantoic fluid is harvested 48h after inoculation and the chick embryo passage is continued, 5 generations of chick embryo allantoic fluid is blindly transmitted and harvested, the chick embryo allantoic fluid of 5 th generation is named rH 120F 5, and the chick embryo allantoic fluid is split-packed and stored at-80 ℃ after being filtered by a 0.22 mu M filter membrane.
Experimental example 1
Identification of rescue Virus
RT-PCR detection
200. Mu.L of rH 120F 5 strain and rH 120-delta 5a/H9HA F5 virus solution are taken, viral RNA is extracted according to the instruction of an Axyprep humoral virus DNA/RNA small extraction kit, the extracted RNA is dissolved in 40. Mu.L of RNase-free TE buffer, and 10. Mu.L of RNA is taken to remove DNA according to the instruction of Recombinant DNase I (RNase-free) (Takara).
Using the RNA as a template, and a primer
M-F：5’-CCTAAGAACGGTTGGAAT-3’；
M-R：5’-TACTCTCTACACACACAC-3’；
S1-F：5’-AAGACTGAACAAAAGACCGACT-3’；
S1-R：5’-CAAAACCTGCCATAACTAACATA
-3’5ab-F：ACCACCTACACTACTTACTTG；
5ab-R：ATTATCTGTGTGTTCCTCACAAG
PrimeScript as one-step RT-PCR kit TM The One Step RT-PCR Kit Ver.2 instructions amplified the M, S and 5ab genes. The PCR products were subjected to 1% agarose gel electrophoresis to observe the amplification results, which are shown in FIG. 6, and sent to Shanghai Bioengineering company for sequencing.
Indirect Immunofluorescence (IFA)
In order to identify the expression of the exogenous proteins of the recombinant viruses, the rescued rH 120F 5 and the recombinant viruses H120-delta 5a/H9HA are inoculated onto a freshly prepared single-layer primary CK according to the 100MOI virus amount, and simultaneously, a null CK control is arranged, the cell culture solution before inoculation is replaced by a DMEM culture solution containing 2 percent of fetal bovine serum, and the culture solution is cultured at 37 ℃ and 5 percent CO 2 Is provided. And (3) inoculating the virus for 36H, and detecting the exogenous gene expression of the rescued recombinant virus H120-delta 5a/H9HA by indirect Immunofluorescence (IFA). The cell culture medium was aspirated and washed twice with hot PBS; fixing with 4% paraformaldehyde at room temperature for 15-20min; PBST washes cells 3 times, 5 min/time; punching: incubating with 0.4% -0.5% Triton-100 (PBS), i.e. Triton-100:pbs=0.4:100, for 15-20min; PBST washes cells 3 times, 5 min/time; closing: blocking with 5% BSA at room temperature for 30min or overnight at 4deg.C; primary antibody (monoclonal antibody against H9 subtype HA gene), incubated for 1H at room temperature; PBST washes cells 3 times, 5 min/time; light-shielding secondary antibody (FITC labeled goat anti-rabbit IgG), and incubating for 1h at room temperature; light-shielding PBST washing for 3 times each for 5minA/times; light-shielding, nuclear staining: DAPI: pbs=1:200, incubate for 15min; light was protected from PBST and washed 3 times, 5 min/time. The result of photographing with a fluorescence microscope is shown in FIG. 7.
rH120-delta 5a/H9HA virus growth curve assay
rH120-delta 5a/H9HA and rH120 were diluted with physiological saline and inoculated with 30 SPF chick embryos, 100EID 50/embryo, each at 10 days old via the allantoic cavity. And respectively harvesting virus allantoic fluid of 5 chick embryos at 6h,12h,24h,36h and 48h after inoculation, mixing, split charging, and freezing at-80 ℃. The Reed-Muench method was used to determine the viral EID50 at various time points.
Claims (2)
1. A method for preparing infectious bronchitis virus rH120-delta 5a/H9HA for expressing HA genes, which is characterized by comprising the following steps:
s1: constructing a viral plasmid vector p15A-H120 of an infectious bronchitis virus vaccine strain H120 by utilizing a plasmid vector p 15A;
s2: amplifying an amp-ccdB bidirectional screening marker gene expression cassette containing a homology arm by taking a plasmid pBR322-kanR-amp-ccdB-rpsLneo as a template;
s3: transferring the viral plasmid vector p15A-H120 in the step S1 and the amp-ccdB bidirectional screening marker gene expression cassette in the step S2 into competent GBred gyrA462 somatic cells together, and carrying out homologous recombination to obtain a recombinant plasmid p15A-H120-amp-ccdB;
s4: transferring a gene expression cassette containing an HA gene shown as SEQ ID No.1 and the recombinant plasmid p15A-H120-amp-ccdB obtained in the step S3 into competent GBdir E.coli together, and screening to obtain recombinant plasmid p15A-H120-HA;
s5: constructing helper plasmid pVAX1-H120N, transfecting BSR-T7 cells with the recombinant plasmid p15A-H120-HA and the helper plasmid pVAX1-H120N in the step S4 together, collecting the transfected supernatant, transferring the supernatant into chick embryos, and carrying out passage to obtain the infectious bronchitis virus rH 120-delta 5A/H9HA for expressing the HA gene;
the infectious bronchitis virus rH120-delta 5a/H9HA is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: v201931.
2. Use of the method of claim 1 for the preparation of infectious bronchitis virus expressing HA gene of H9N2 avian influenza virus.
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