CN105002146A - Turkey herpes virus strain rHVT-H9HA and its construction method - Google Patents

Abstract

The invention relates to a recombinant turkey herpes virus (HVT) rHVT-H9HA expressing H9 subtype avian influenza virus (AIV) hemagglutinin protein and a construction method thereof. The preservation number of the vaccine strain rHVT-H9HA is CGMCC? No: 10907. The GFP expression cassette was isolated from the vector pEGFP-C1 and inserted into the HVT genome to obtain recombinant virus rHVT-GFP. Through homologous recombination again, the HA gene of the H9N2 subtype AIV epidemic strain A/Chicken/Jiangsu/WJ57/2012 was replaced with the GFP gene of the recombinant virus rHVT-GFP, and the recombinant virus without fluorescence was selected to obtain a stable expression of the H9 subtype AIV? The turkey herpesvirus strain rHVT-H9HA of the HA gene. The recombinant virus strain has low cost, good safety and long immunity period, is suitable for large-scale production of vaccines, and can be used to manufacture vaccines.

Description

Turkey herpes virus strain rHVT-H9HA and its construction method

technical field

The invention relates to the field of recombinant virus vaccines, more specifically, the invention relates to a turkey herpes virus strain rHVT-H9HA expressing the H9 subtype HA gene of avian influenza, which is used for the development of the vaccine. The invention also provides a method for developing the recombinant turkey herpes virus vaccine strain.

Background technique

Among the many pathogens that cause poultry diseases, avian influenza virus (AIV) and Marek's disease virus (MDV) occupy an important position. Prevention and control of outbreaks of avian influenza and Marek's disease in poultry is of great significance to animal health, public health and the world economy.

Avian influenza (Avian influenza, AI) is a viral infectious disease that seriously endangers the development of the global poultry industry. AIV belongs to the genus of influenza virus A in the Orthomyxoviridae family. According to the difference in pathogenicity to chickens, AIV can be divided into low pathogenicity AIV and high pathogenicity AIV. Among them, the low-pathogenic H9 subtype AIV can not only infect poultry, resulting in decreased egg production, immunosuppression and high mortality after mixed infection with other pathogens, but also can infect humans, pigs and other mammals. It is worth noting that viruses of this subtype frequently provide internal genes for the emergence of new influenza viruses. For example, the six internal segments of H5N1, H7N9, and H10N8 that infect humans all come from H9N2, so they have important public health significance. At present, avian influenza vaccines are mainly whole-virus inactivated vaccines. Although the immune protection effect is good, there are still problems such as high production cost, inconvenient use, interference with serological monitoring, great interference by maternal antibodies during use, and only The induction of humoral immunity and potential safety hazards may cause a series of problems such as artificial dispersal of poison. There is currently no live vaccine against H9 subtype AIV on the market. Therefore, it is of great practical significance to develop a new and efficient next-generation vaccine for the prevention and control of H9 subtype AIV.

Herpesvirus of Turkey (HVT) is a typical non-pathogenic alpha herpes virus. Since HVT is genetically and serologically related to chicken MDV, the HVT Fc-126 strain has been widely used as a MD vaccine since 1970. The vaccine has many advantages such as no tumorigenesis, good protective effect, persistent existence in the body, freeze-drying, easy storage and transportation, etc., and has played an important role in the prevention and control of MD. In recent years, the research on HVT as a viral vector has made a lot of progress, because HVT has many unique advantages compared with other poultry virus vectors, including: HVT is non-pathogenic to chickens and other animals, and has good safety; it can be used in animals Persistent existence, which is conducive to the continuous expression of foreign genes, and stimulates the body to produce higher antibody levels; HVT can cause high-intensity cell-mediated immune responses, early immune emergence, long duration, and lifelong immunity can be achieved once inoculated; HVT Recombinant vaccines have low production costs, can be freeze-dried, and are easy to store and transport; HVT has strong cell-binding properties, and the virus spreads between cells, so as a recombinant vaccine, it can break through the interference of maternal antibodies, etc. [Bublot, M., et al., Use of a vectored vaccine against infectious bursaldisease of chickens in the face of high-titred maternally derived antibody. J Comp Pathol, 2007.137 Suppl 1:p.S81-4.]. At present, various avian disease protective antigens such as infectious bursal virus have been successfully expressed in HVT vectors [Tsukamoto, K., et al., Complete, Long-Lasting Protection against Lethal Infectious Bursal Disease Virus Challenge by a Single Vaccination with an Avian Herpesvirus Vector Expressing VP2 Antigens. Journal of Virology, 2002.76(11): p.5637-5645.]. The Vaxxitex ^R HVT+IBD recombinant vaccine prepared at the above level has obtained the production license and has become a commercialized animal-derived vaccine with herpes virus as the carrier, making HVT the most promising viral vector. The core of its technology uses the principle of homologous recombination: through the homologous exchange of the non-essential region homology arms on both sides of the foreign gene and the corresponding segment of the HVT genome, the foreign gene is introduced into the genome. In 1992, Morgan et al first used homologous recombination method to construct recombinant HVT expressing NDV(F) gene. This recombinant virus can not only resist the attack of lethal MDV, but also prevent Newcastle disease, and its protection rate is close to that of traditional vaccines. [Morgan, RW, et al., Protection of chickens from Newcastle and Marek's diseases with are combinant herpesvirus of turkeys vaccine expressing the Newcastle disease virus fusion protein. Avian Dis, 1992.36(4): p.858-70.]. Later, Gao et al. used the above method to insert the AIV H5N1 (HA) gene into different positions of the HVT genome, and constructed two strains of rHVT recombinant viruses. Vaccine tests showed that both strains of recombinant viruses could resist both AI and MD [Gao, H., et al., Expression of HA of HPAI H5N1 Virus at US2 Gene Insertion Site of Turkey Herpesvirus Induced Better Protection than That at US10 Gene Insertion Site. PLoS ONE, 2011.6(7): p.e22549.]. In 2012, Gu Xiaobing and others isolated a representative H9N2 subtype AIV epidemic strain A/Chicken/Jiangsu/ _WJ57 /2012 in Wujin, Changzhou. Sex; its HA gene is genetically derived from the A/Duck/HongKong/Y280/97-like subline of the H9N2 subtype AIV Eurasian lineage. Genetic evolution analysis showed that the WJ57 strain was on the popular branch and was representative. Therefore, the present invention uses the HVT Fc-126 strain as a viral vector to construct a recombinant vaccine strain capable of expressing the H9 subtype AIV hemagglutinin protein to cope with the current epidemic of H9 subtype AI and MD.

In the process of recombinant virus construction, screening is an important part of successfully obtaining the target recombinant virus. Because HVT has a strong cell-binding property, it brings great difficulties to the screening of HVT recombinant virus. At present, the most commonly used method is the marker gene insertion method to screen recombinant viruses. However, the presence of marker genes has brought many disadvantages, such as immunizing animals with recombinant viruses carrying the LacZ gene of Escherichia coli. The expression of LacZ gene may interfere with the immune effect of recombinant viruses. ; In addition, immunization of animals with recombinant viruses carrying antibiotic genes will also cause animals to develop drug resistance. Therefore, choosing an effective recombinant virus construction and screening method can not only greatly reduce the workload of screening recombinant viruses, but also lay a technical foundation for obtaining a high-efficiency recombinant virus vaccine.

Contents of the invention

The first purpose of the present invention is to overcome the above-mentioned deficiencies of the existing avian influenza whole virus inactivated vaccines, and to invent a recombinant vaccine strain expressing the H9 subtype AIV hemagglutinin protein with the turkey herpes virus as a carrier for the manufacture of vaccines .

The recombinant turkey herpes virus strain rHVT-H9HA expressing the H9 subtype AIV hemagglutinin protein of the present invention was deposited in the General Microorganism Center of China Committee for Microbial Culture Collection on June 30, 2015, and its preservation number is CGMCC No: 10907.

Second object of the present invention is to invent a kind of construction method of recombinant turkey herpes virus vaccine strain:

The invention utilizes the principle of homologous recombination, separates the GFP expression cassette from the carrier pEGFP-C1 and inserts it into the HVT genome, and obtains the recombinant virus rHVT-GFP expressing the green fluorescent protein through several rounds of screening and purification. Recombinant rHVT-GFP has certain universal properties. The transfer plasmid vector containing other exogenous genes constructed by the same homology arm and rHVT-GFP genomic DNA are co-transfected into cells, and other exogenous genes can be replaced with GFP gene to obtain more Recombinant HVT expressing multiple foreign genes.

Through homologous recombination again, the HA gene of an H9 subtype AIV WJ57 epidemic strain isolated in our laboratory was replaced with the GFP gene in the recombinant virus rHVT-GFP, and the recombinant virus without fluorescence was selected, and after several rounds of screening and purification, the obtained A turkey herpesvirus strain rHVT-H9HA stably expressing H9 subtype avian influenza HA gene.

We applied the principle of homologous recombination to go through the first step of screening. First, we obtained a recombinant HVT with a marker gene, and then performed homologous recombination with a transfer plasmid vector containing other foreign genes constructed with the same non-essential fragment. The source gene replaces the marker gene, and the recombinant HVT with other foreign genes is obtained by using the reverse screening method. The recombinant HVT virus obtained by this method is the closest to the maternal HVT. Except for the expressed target gene, there is no other foreign gene, which overcomes many disadvantages brought by the expression of the marker gene, and improves the development and production of recombinant turkey herpes virus vaccine. Development value; at the same time, it also greatly facilitates people to obtain various recombinant HVTs expressing foreign genes, which provides new ideas for the research and development of new vaccines.

The present invention comprises the following specific steps:

1) Amplification and cloning of DNA fragments

PCR was used to amplify the HA of the H9 subtype AIV WJ57 strain, the GFP in the plasmid pEGFP-C1 and the sequences on both sides of the foreign gene insertion site of the genome of the HVT Fc-126 strain, and directionally clone these DNA fragments into the plasmid vector , to obtain recombinant plasmids pHA, pEGFP, pUP, pDOWN.

2) Construction of recombinant transfer vector

① Construction of intermediate plasmid pCUS2

First, the recombinant plasmids pUP and pDOWN obtained above were subjected to a series of digestion and ligation reactions to construct the intermediate plasmid pCUS2.

②Construction of transfer vector containing EGFP marker gene

First, the recombinant plasmids pEGFP and pCUS2 obtained above were subjected to a series of digestion and ligation reactions to construct the transfer vector pCMGFP containing the EGFP marker gene.

③Construction of transfer vector containing HA target gene

First, the recombinant plasmids pHA, pCUS2, and pCMGFP obtained above were subjected to a series of enzyme digestion and ligation reactions to construct the transfer vector pCMHA containing the HA target gene.

3) Construction and purification of recombinant virus

① Construction and purification of recombinant virus rHVT-GFP expressing green fluorescent protein:

Genomic DNA and the DNA of the transfer vector pCMGFP were extracted from the HVT Fc-126 strain, and co-transfected into CEF cells by the calcium phosphate method. Homologous recombination occurred between the two, and the expression cassette containing the GFP gene was inserted into the HVT genome. Fluorescent plaques are picked, and after several rounds of screening and purification, it is confirmed that all plaques are fluorescent. A recombinant virus strain rHVT-GFP with screening marker-green fluorescence was obtained;

② Construction and purification of recombinant virus rHVT-H9HA expressing H9 subtype avian influenza HA protein:

Genomic DNA and the DNA of the transfer vector pCMHA were extracted from the above-mentioned purified recombinant virus rHVT-GFP, and the calcium phosphate method was used to co-transfect the cell CEF. Homologous recombination occurred between the two, and the expression cassette containing the HA gene was inserted into the HVT genome. . Virus plaques without fluorescence were picked, and after several rounds of screening and purification (until all virus plaques had no fluorescence), the recombinant virus turkey herpesvirus strain rHVT-H9HA was obtained.

Description of drawings

Fig. 1 Schematic diagram of the construction of recombinant plasmid pUS2.

Fig. 2 EcoRI digestion identification of transfer vector plasmids pCMGFP and pCMHA.

Fig. 3 Construction model diagram of recombinant virus rHVT-GFP and rHVT-H9HA.

Fig. 4 Fluorescence pictures of recombinant virus rHVT-GFP.

Fig. 5 The picture of the recombinant virus rHVT-H9HA without fluorescence.

Fig. 6 Detection of exogenous gene expression in CEF infected by recombinant virus rHVT-H9HA.

The virus strain rHVT-H9HA was preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on June 30, 2015 (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), named : Recombinant turkey herpes virus strain rHVT-H9HA; its preservation number CGMCC No: 10907.

Detailed ways

Step 1: Amplification of DNA Fragments

Biomaterial preparation:

HVT Fc-126 strain [Hu Chuanwei, Peng Daxin, Zhang Rukuan, Liu Xiufan. Immunological efficacy of recombinant fowl pox virus expressing chicken Marek's disease virus gB gene and turkey herpes virus bivalent freeze-dried vaccine[J]. Chinese Journal of Veterinary Medicine, 2006, 01:11‐13.], preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University.

The positive cloning plasmid pPWJM containing the HA gene of H9N2 subtype AIV epidemic strain WJ57 [Ding Pingyun, Liu Xiufan et al. Construction and rescue of H9 subtype avian influenza and Newcastle disease recombinant vaccine strains[J]. China Poultry, 2013, 16: 16-20.], constructed and preserved by the Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University.

pEGFP-C1 eukaryotic expression plasmid: purchased from BD Biosciences Clontech; pCR2.1 vector: purchased from Invitrogen; High fidelity DNA polymerase, T4 DNA ligase, Agarose Gel DNA Extraction Kit were purchased from Roche; plasmid extraction kit (QIAprep Spin MiniPrep Kit) is a product of QIAGEN; the rest of the conventional reagents are domestic analytical grade.

1) Amplification of HA of H9 subtype AIV WJ57 strain

Design a pair of DNA primers with Nhe I and Kpn II restriction sites at both ends, the sequence is:

HA-F: 5'-CATgctagcATGGAGACAGTATCACTAATAACT-3'(Nhe I) (SEQ ID NO.1)

HA-R: 5'-GGCtccggaTTATATACAAATGTTGCATCTG-3'(Kpn II) (SEQ ID NO.2)

PCR amplification of the full-length fragment of the HA gene (50 μL reaction system): use the plasmid pPWJM containing the HA gene as a template, suck it into a 0.5 mL PCR reaction tube, and amplify the HA gene of the WJ57 strain with primers HA-F and HA-R, The specific operation method is as follows:

PCR reaction cycle parameters: pre-denaturation at 94°C for 3min; 30s at 94°C, 45s at 56°C, 2min at 72°C, 10min at 72°C after 20 cycles, and storage at 4°C.

2) Amplification of the screening marker gene GFP expression cassette

Design a pair of DNA primers with EcoR V and Not I restriction sites at both ends, the sequence is:

GFP-F: 5'-CCCgatatcTAGTTATTAATAGTAATCAATTACGG-3'(EcoR V) (SEQ ID NO.3)

GFP-R: 5'-AAAgcggccgcGTTAAGATACATTGATGAGTT-3'(Not I) (SEQ ID NO.4)

PCR reaction system: the specific operation method is as follows:

PCR reaction cycle parameters: 94°C pre-denaturation for 4min; 94°C for 30s, 55°C for 45s, 72°C for 1min/kb extension, after 20 cycles, 72°C for 10min, and 4°C for storage.

3) Amplification of sequences on the left and right sides of the foreign gene insertion site in the HVT genome

DNA Preparation of HVT Infected Cells

The primary and secondary CEF cells were prepared according to conventional methods. The HVT Fc-126 strain was inoculated into the secondary CEF cultured in T75 cell culture flasks and cultured at 37°C. 80% of the cells produced typical cytopathic changes. The cells were harvested and the HVT genome was extracted according to traditional methods . The specific operation steps are as follows: Remove the medium, wash once with PBS (pH7.2), discard the PBS, add 5mL PK solution (containing 200mM Tris-HCl, pH8.0; 1.5M NaCl; 20mM EDTA, pH8.0 ; 5% SDS; 1mg proteinase K), let stand at room temperature for 10min, transfer the lysed cells to a 50mL centrifuge tube, and place in a 37°C incubator for 1.5h; add 1/2 volume of phenol, gently invert to mix, and use 2min; add 1/2 volume of chloroform/isoamyl alcohol (24:1), shake gently, centrifuge at 8000r/min for 10min; take the upper aqueous phase into a clean centrifuge tube, add 1 volume of chloroform/isoamyl alcohol , shake gently, and centrifuge at 8000r/min for 10min; take the upper aqueous phase into a clean centrifuge tube, add 1/10 volume of 3mol/L sodium acetate (pH5.2) and 2 volumes of 95% ethanol, gently Mix well, place at -20°C, precipitate for 30min, centrifuge at 5000r/min for 5min, discard the supernatant; add 10mL of 70% ethanol to wash the precipitate, centrifuge at 5000r/min for 5min, discard the supernatant, and dissolve the precipitate in TE with pH7.0 after natural drying , Measure the DNA content, dispense into sterile finger tubes, and store at 4°C for later use.

Design DNA primers UP-F and UP-R to amplify the UP sequence, with Hind III and Sac I restriction sites at both ends, the sequence is:

UP-F: 5'-CCCaagcttATCCTTACTAGCGCCGATT-3' (Hind III) (SEQ ID NO.5)

UP-R: 5'-TTTgagctcGGGCCATTCCATGTGAGATACAAAC-3'(Sac I) (SEQ ID NO.6)

Design DNA primers DOWN-F and DOWN-R to amplify the DOWN sequence, with Not I and Xba I restriction sites at both ends, the sequence is:

DOWN-F: 5'-AAAAAAgcggccgcCCGGAACCGGGCATACCACT-3'(Not I) (SEQ ID NO.7)

DOWN-R: 5'-TTTgagctcGGGCCATTCCATGTGAGATACAAAC-3'(Xba I) (SEQ ID NO.8)

PCR reaction system: the specific operation method is as follows:

PCR reaction cycle parameters: 94°C pre-denaturation for 4min; 94°C for 30s, 60°C for 45s, 72°C for 1min/kb extension, after 25 cycles, 72°C for 10min, and 4°C for storage.

The 139401nt-140408nt region of the HVT genome was amplified with primers UP-F and UP-R, and the 140651nt-141657nt region of the genome was amplified with primers DOWN-F and DOWN-R.

Step 2: Cloning of DNA Fragments

After electrophoresis, the RT-PCR and PCR products were cut into gels containing fragments of the desired size, recovered and purified with the Agrose Gel DNA Extraction Kit, connected to the pCR-2.1T vector, transformed into E. After enzyme digestion and identification were correct, they were sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing, and the positive plasmids with correct sequence determination were named pHA, pEGFP, pUP, and pDOWN respectively.

Step 3: Construction of recombinant transfer vector

1. Construction of intermediate vector pCUS2

The plasmid pUP was digested with Hind III and Sac I, the insert fragment was extracted, and then cloned into the Hind III and Sac I sites of the plasmid pCR2.1 vector to obtain pCR2.1-UP. The plasmid pDOWN was digested with Not I and Xba I, the insert fragment was also extracted, and then cloned into the Not I and Xba I sites of the vector pCR2.1-UP, and the intermediate plasmid was constructed and identified by PCR. The positive clone was named pCUS2 (Fig. 1).

2. Construction of gene transfer vector containing GFP marker

The plasmid pEGFP was digested with EcoR V and Not I, the insert was extracted, and then cloned into the EcoRV and Not I sites of the intermediate plasmid pCUS2, the constructed recombinant plasmid was identified by restriction enzyme digestion and PCR, and the positive clone was named pCMGFP (Fig. 2 ).

3. Construction of HA-containing target gene transfer vector

Using the pCMGFP plasmid cut by Nhe I and Kpn II as the carrier, the plasmid pHA was digested by Nhe I and Kpn II to replace the corresponding sequence GFP gene in the transfer vector pCMGFP, and the recombinant plasmid constructed was identified by enzyme digestion and PCR, and the positive clone was named is pCMHA (Figure 2).

Step 4: Construction and purification of recombinant virus

1. Construction and purification of recombinant virus rHVT-GFP

(1) Preparation of HVT-infected cell DNA

The primary and secondary CEF cells were prepared according to conventional methods, and 10 ⁵ plaque-forming units (PFU) of the HVT Fc-126 strain were inoculated in the secondary CEF of each bottle of T75 cell culture flask, cultured for 3-5 days, and 80% of the cells produced typical The cells were harvested, and the HVT genome was extracted according to the traditional method, and the specific operation steps were the same as above.

(2) Transfer vector pCMGFP and HVT genomic DNA co-transfect CEF

The transfer vector pCMGFP plasmid DNA was extracted with QIAprep Spin MiniPrep Kit (QIAGEN). CEFs were prepared according to conventional methods, and the secondary CEFs were prepared 1 hour before the start of the transfection experiment. 3×10 ⁶ cells were spread on each 60 mm culture dish, and cultured at 37° C. and 5% CO 2 .

Add the following reagents in sequence to a sterile finger tube:

Sterile ultrapure water 194μL

HVT DNA 5 μg

Transfer vector pCMGFP plasmid 1 μg

TE Make up to 25μL

At the same time, it is necessary to set up an HVT control group without adding the transfer vector pCMGFP plasmid, and the system is as follows:

Sterile ultrapure water 194μL

HVT DNA 5 μg

TE Make up to 25 μL

After mixing the above system, slowly add 31 μL 2mol/L CaCl ₂ solution and 250 μL 2xHBSP solution (42mmol/L HEPES, 1.4mmol/L Na ₂ HP04, 274mmol/L NaCl, 5mmol/L KCI, 6mmol of pH 7.05) from the bottom of the tube /L glucose), gently blow out several bubbles from the bottom of the tube with a pipette, and let stand at room temperature for 30min to form a CaP0 ₄ -DNA mixture. Add 500 μL of CaP0 ₄ -DNA complex to each 60 mm culture dish covered with secondary CEF cells, shake gently, and continue to incubate for 4 hours. Discard the medium and add 2 mL of glycerol shock to each cell culture dish.

The system of 2.5ml glycerol shock solution is as follows:

Sterile ultrapure water 0.9mL

Glycerin 0.35mL

2xHBSP 1.25mL

After mixing the above system and acting for 1 min, discard the shock solution, wash the cells with maintenance solution (V) medium, discard the washing solution, add growth medium (G) medium containing 4% fetal bovine serum, and continue to culture for 5-7 days Observed with an inverted fluorescence microscope.

(3) Screening and purification of recombinant virus rHVT-GFP

The transfected CEFs were observed with an inverted fluorescence microscope, and virus plaques with green fluorescence were marked. Discard the medium, scrape the virus plaques with a pipette with a little trypsin, transfer to 10mL growth medium for dilution, and inoculate each virus plaque on a 96-well plate that has been plated with sub-generation CEF cells, and inoculate each well 100 μL of diluted virus plaques were cultured at 37°C and 5% CO2. Repeat the above steps until all the virus plaques on the 96-well cell culture plate have green fluorescence, and the cells that make up each virus plaque have fluorescence, indicating that the recombinant virus is completely purified, and the recombinant virus is named rHVT- GFP (FIGS. 3, 4).

2. Construction and purification of recombinant virus rHVT-H9HA

(1) Preparation of rHVT-GFP infected cell DNA

The primary and secondary CEF cells were prepared according to conventional methods, inoculated 10 ⁵ plaque-forming units (PFU) of the recombinant rHVT-GFP virus strain in the secondary CEF of each bottle of T75 cell culture flask, cultured for 3-5 days, and equal to 80% of the cells Harvest the cells after producing typical cytopathic changes, and extract the HVT genome according to the traditional method, and the specific operation steps are the same as above.

(2) Co-transfection of CEF cells with transfer vector pCMHA and rHVT-GFP genomic DNA

The transfer vector pCMHA plasmid DNA was extracted with QIAprep Spin MiniPrep Kit (QIAGEN). CEFs were prepared according to conventional methods, and the secondary CEFs were prepared 1 hour before the start of the transfection experiment. 3×10 ⁶ cells were spread on each 60 mm culture dish, and cultured at 37° C. and 5% CO 2 .

Add the following reagents in sequence to a sterile finger tube:

At the same time, it is necessary to set up an HVT control group without adding the transfer vector pCMGFP plasmid, and the system is as follows:

Sterile ultrapure water 194μL

rHVT-GFP DNA 5 μg

TE Make up to 25 μL

After mixing the above system, slowly add 31 μL 2mol/L CaCl ₂ solution and 250 μL 2xHBSP solution (42mmol/L HEPES, 1.4mmol/L Na ₂ HP04, 274mmol/L NaCl, 5mmol/L KCI, pH7.05 6mmol/L glucose), gently blow out several bubbles from the bottom of the tube with a straw, and let it stand at room temperature for 30min to form a CaP0 ₄ -DNA mixture. Add 500 μL of CaP0 ₄ -DNA complex to each 60 mm culture dish covered with secondary CEF cells, shake gently, and continue to incubate for 4 hours. Discard the medium and add 2 mL of glycerol shock to each cell culture dish.

The system of 2.5ml glycerol shock solution is as follows:

Sterile ultrapure water 0.9mL

Glycerin 0.35mL

2xHBSP 1.25mL

After mixing the above system and acting for 1 min, discard the shock solution, wash the cells with maintenance solution (V) medium, discard the washing solution, add growth medium (G) medium containing 4% fetal bovine serum, and continue to culture for 5-7 days Observed with an inverted fluorescence microscope.

(3) Screening and purification of recombinant virus rHVT-H9HA

The transfected CEFs were observed with an inverted fluorescent microscope, and virus plaques without green fluorescence were marked. Discard the medium, scrape the virus plaques with a pipette with a little trypsin, transfer to 10mL growth medium (G) medium for dilution, and inoculate a 96-well plate with subcultured CEF cells for each virus plaque, Each well was inoculated with 100 μL of diluted virus plaques and cultured at 37°C and 5% CO2. Repeat the above steps until all the virus plaques on the 96-well cell culture plate do not have green fluorescence, and the cells that make up each virus plaque do not have fluorescence, indicating that the recombinant virus is completely purified, and the recombinant virus is named as rHVT-H9HA (Fig. 3, 5).

Step 5: Passaging and Identification of Recombinant Viruses

(1) After rHVT-GFP was uploaded to CEF cells for 20 generations, observed under a fluorescent microscope, there was no loss of fluorescence, indicating that the recombinant virus can be inherited stably.

(2) rHVT-H9HA was uploaded to CEF cells for 20 generations, and was identified by PCR every five generations. The identification results showed that the HA genes of the 5th, 10th, 15th, and 20th generations were all successfully inserted into the HVT genome, and The sequence is correct.

(3) Detection of rHVT-H9HA expressing H9HA

Indirect immunofluorescence assay (IFA): Infect secondary CEF cells with rHVT-H9HA, and form viral plaques in 3-5 days; fix the cells with cold methanol for 30 minutes, wash 3 times with PBS; add working concentration of anti-AIVHA polyclonal antibody (H9 Serum), acted at 37°C for 1 hour, washed 2-3 times with PBS; added anti-chicken IgG/lgM fluorescent monoclonal antibody at working concentration, acted at 37°C for 1 hour, washed 2-3 times with PBS; observed under a fluorescent microscope, the results It was shown that all cells making up the viral plaques were fluorescent (Fig. 6).

Claims (4)

1. express the recombinant herpesvirus of turkeys strain rHVT-H9HA of H9 subtype avian influenza virus (AIV) hemagglutinin, it is characterized in that: the HA albumen of energy stably express H9 hypotype AIV, its preserving number CGMCC No:10907.

2. express the construction process of recombinant herpesvirus of turkeys (HVT) rHVT-H9HA of H9 hypotype AIV hemagglutinin as claimed in claim 1, it is characterized in that: application homologous recombination principle, from pEGFP-C1 carrier, be separated GFP expression cassette is inserted in HVT genome, obtains the recombinant virus rHVT-GFP of expressing green fluorescent protein through screening and purifying; By homologous recombination again HA gene replaced the GFP gene in recombinant virus rHVT-GFP, select the recombinant virus not with fluorescence, through screening and purifying, obtain the herpes turkey virus strain rHVT-H9HA of stably express H9 subtype avian influenza HA gene.

3. express the construction process of the recombinant herpesvirus of turkeys strain rHVT-H9HA of H9 hypotype AIV hemagglutinin as claimed in claim 2, it is characterized in that comprising following concrete steps:

1) amplification of DNA fragmentation and clone:

Build positive plasmid pHA: from plasmid pPWJM, amplify gene fragment HA with primer HA-F, HA-R, be connected with pCR2.1 carrier by gene fragment HA, obtain positive plasmid pHA;

Build positive plasmid pEGFP: make template with pEGFP-C1 plasmid, amplify GFP expression cassette with primer GFP-F, GFP-R, be connected with pCR2.1 carrier by gene fragment GFP expression cassette, obtain positive plasmid pEGFP;

Build positive plasmid pUP, pDOWN: clone gene fragment UP, DOWN from the 139401nt ~ 140408nt of HVT Fc-126 pnca gene group, 140651nt ~ 141657nt position respectively, gene fragment UP, DOWN are connected with plasmid pCR2.1 carrier, obtain positive plasmid pUP, pDOWN;

The above-mentioned primer sequence for increasing is as follows:

HA-F：5’-CATgctagcATGGAGACAGTATCACTAATAACT-3’

HA-R：5’-GGCtccggaTTATATACAAATGTTGCATCTG-3’

GFP-F：5’-CCCgatatcTAGTTATTAATAGTAATCAATTACGG-3’

GFP-R：5’-AAAgcggccgcGTTAAGATACATTGATGAGTT-3’

2) structure of recombinant transfer vector

The structure of middle interstitial granules pCUS2: plasmid pUP Hind III and Sac I enzyme are cut, extracts Insert Fragment, on directed cloning to the Hind III and Sac I site of pCR2.1 carrier, obtains pCR2.1-UP; Plasmid pDOWN Not I and Xba I enzyme are cut, extracts Insert Fragment equally, on subclone to the Not I and Xba I site of carrier pCR2.1-UP, obtain pCUS2;

Structure containing GFP marker gene transfer vector: plasmid pEGFP EcoR V and Not I enzyme are cut, extracts Insert Fragment, on subclone to the EcoR V and Not I site of middle interstitial granules pCUS2, obtains universal transfer plasmids pCMGFP;

Structure containing HA goal gene transfer vector: plasmid pHA, for carrier, is cut sequence GFP gene corresponding in displacement transfer vector pCMGFP through Nhe I and Kpn II enzyme, obtains transfer vector pCMHA by the pCMGFP plasmid cut with Nhe I and Kpn II enzyme;

3) structure of recombinant virus and purifying

1. the structure of the recombinant virus rHVT-GFP of expressing green fluorescent protein and purifying:

Extract the DNA of genomic dna and transfer vector pCMGFP from HVT Fc-126 strain, adopting calcium phosphate method to carry out cotransfection chick embryo fibroblast (CEF), there is homologous recombination in both, is inserted in HVT genome by the expression cassette containing GFP gene; Picking, with the virus plaques of fluorescence, through the screening of number wheel and purifying, until all virus plaques are all with fluorescence, obtains the recombinant virus rHVT-GFP of band selection markers-green fluorescence;

2. structure and the purifying of the recombinant virus rHVT-H9HA of H9 hypotype AIV HA albumen is expressed:

Extract the DNA of genomic dna and transfer vector pCMHA from the recombinant virus rHVT-GFP that above-mentioned purifying is good, adopting calcium phosphate method to carry out cotransfection cell CEF cell, there is homologous recombination in both, is inserted in HVT genome by the expression cassette containing HA gene; Picking, without the virus plaques of fluorescence, through the screening of number wheel and purifying, until all virus plaques are not with fluorescence, obtains herpes turkey virus strain rHVT-H9HA.

4. express the construction process of the recombinant herpesvirus of turkeys strain rHVT-H9HA of H9 hypotype AIV hemagglutinin according to claim 3, it is characterized in that step 1,

The primer cloning gene fragment UP from HVT Fc-126 pnca gene group is:

UP-F：5’-CCCaagcttATCCTTTACTAGCGCCGATT-3’

UP-R：5’-TTTgagctcGGGCCATTCCATGTGAGATACAAAC-3’

The primer cloning gene fragment DOWN from HVT Fc-126 pnca gene group is:

DOWN-F：5’-AAAAAAgcggccgcCCGGAACCGGGCATACCACT-3’

DOWN-R：5’-TTTgagctcGGGCCATTCCATGTGAGATACAAAC-3’。