CN112852755A - Preparation and application of inactivated vaccine of gE/gI gene deletion strain of pseudorabies virus FJ-2012 strain - Google Patents

Abstract

The invention relates to a gene deletion strain of pseudorabies virus FJ-2012 strain gE/I, which is named as a pseudorabies virus FJ-2012 delta gE/gI strain with the preservation number of CCTCC NO: V202025. According to the invention, a gE/gI gene deletion strain of a new pseudorabies virus variation FJ-2012 strain is used as a vaccine seed virus, formaldehyde is inactivated through amplification culture and then the inactivated vaccine is prepared into an inactivated vaccine immune sheep with different adjuvants, the inactivated vaccine is attacked by a parental virulent strain FJ-2012 strain after being immunized for 4 weeks, the inactivated vaccine provides complete immune protection effect on the immune sheep, and the method is suitable for identifying and diagnosing wild virus infection or vaccine immunity by using a general gE ELISA identification and diagnosis kit at present.

Description

Preparation and application of inactivated vaccine of gE/gI gene deletion strain of pseudorabies virus FJ-2012 strain

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to preparation and application of a pseudorabies virus FJ-2012 strain gE/gI gene deletion strain inactivated vaccine.

Background

Pseudorabies (PR), also known as Aujeszky's Disease (AD), is an acute infectious Disease of various domestic and wild animals caused by Pseudorabies virus (PRV). Pseudorabies virus belongs to herpesviridae, sub-family of alpha-herpesviridae and genus varicella herpesviridae, can infect various animals such as pigs, cows, sheep, mice, rabbits, cats, dogs and the like, and pigs are the only species that can survive PRV infection and therefore are also the storage host of the virus. The Halloween and the Hongshang respectively report that porcine pseudorabies occurs in suckling pigs in China in the fifth and sixty years of the last century, but PR is massively developed for the first time in China in the 90 th year of the 20 th century and is expressed as classical PR symptoms, sow abortion and piglet neurological symptoms; PR is in large-scale outbreak at the beginning of 21 st century in China, and is expressed as abortion storm, but since 90 s in 20 th century, Barthak-61 vaccine strains are commonly used in large-scale pig farms and are well controlled; since 10 months in 2011, the field virus positive rate of a high-density PRV immune pig farm is high, and an adult pig and a fattening pig die suddenly, PR shows the third outbreak in China, and the PRR is coiled in China, rapidly spread to other provinces and cities in China, and cause serious influence on the pig industry in China, and is proved to be caused by new variant PRV through etiology research of a plurality of research teams in China.

gE is one of important virulence genes of PRV, PRV lacking gE only can be used for local nerve infection, the invasion capability to the nervous system is reduced, and gE is the first marker gene for detecting PRV, and a serological detection method aiming at a gE antibody is very mature and is a classical serological method for distinguishing wild virus infection or vaccine immunity. The great manpower, material resources and financial resources are invested in the disease in part of European and American countries, and the disease is purified and eliminated in domestic pigs by a gE-ELISA differential diagnosis technology. For years, the technology of gE-deleted attenuated live vaccine immunization, wild virus monitoring, purification and the like is generally adopted in China, the disease is also effectively controlled, the morbidity and the epidemic situation of the disease tend to be stable, but from 2011 for 10 months, pseudorabies outbreak and epidemic occur again in Bartha-K61 immunized large-scale pig farms in most areas of China, and the variant PRV separated after 2011 has lethality for fattening pigs aged 85 days, and the method is mainly characterized in that: (i) pregnant sows immunized with Bartha-K61 vaccine developed high miscarriage rates; (ii) growing pigs develop central nervous system disorders and have high mortality; (iii) the serum has high gE positive rate. All new variant ultrastrong PRV strains isolated after 2011 had significant variation in genomic sequence compared to european and american strains or chinese classical strains isolated before 2000. In recent years, a large number of new PRV strains including HNX, ZJ01, HeN1, SMX, TJ, JS, HNB, HN1201, FJ-2012 and the like are separated from different regions of China, and the existing vaccines cannot provide good protection for the new variant strains, so that a new PR vaccine is urgently needed to be developed by the new variant PRV super-virulent strains so as to better control and prevent PR.

Therefore, there is a need for new vaccines that are safe and effective against new variant PRV infections and that have differential diagnosis. According to the invention, a gE/gI gene deletion strain of a new pseudorabies virus variation FJ-2012 strain is used as a vaccine seed virus, formaldehyde is inactivated through amplification culture and then the inactivated vaccine is prepared into an inactivated vaccine immune sheep with different adjuvants, the inactivated vaccine is attacked by a parental virulent strain FJ-2012 strain after being immunized for 4 weeks, the inactivated vaccine provides a complete immune protection effect on the immune sheep, and the inactivated vaccine is suitable for the identification and diagnosis of wild virus infection or vaccine immunity through a currently universal gE ELISA identification and diagnosis kit, so that the FJ-2012 gE/gI gene deletion strain is suitable for being used as an inactivated vaccine candidate seed virus for preventing the infection of the new variation PRV supervirulent strain.

Disclosure of Invention

The invention aims to provide a preparation method and application of a pseudorabies virus FJ-2012 strain gE/gI gene deletion strain inactivated vaccine.

In order to achieve the purpose, the invention adopts the following technical scheme:

a pseudorabies virus FJ-2012 strain gE/I gene deletion strain is named as a pseudorabies virus FJ-2012 delta gE/gI strain, is preserved in China center for type culture collection (CCTCC NO: V202025) at 8 months 4 in 2020, and has a preservation number of V202025, and the China Center for Type Culture Collection (CCTCC) address is Wuhan university in China.

The preparation method of the gE/I gene deletion strain of the pseudorabies virus FJ-2012 strain comprises the following steps:

(1) sequentially inserting upstream and downstream homologous fragments of a gE/I deletion position of an FJ-2012 strain into a plasmid pUC19 to construct pUC19: H1: H2, then inserting an EGFP expression cassette to construct a homologous recombination transfer plasmid pUC19: H1: EGFP: H2, and then introducing a BAC sequence into pUC19: H1: EGFP: H2 to form a homologous recombination transfer plasmid pUC19: H1: EGFP: BAC 2 capable of constructing a PRV artificial chromosome;

(2) extracting infectious genome DNA of FJ-2012 strain, co-transfecting cells with transfer plasmids pUC19, H1, EGFP, BAC, H2, and screening recombinant virus rPRV FJ-2012-delta gE/gI and EGFP containing fluorescent marker gE/I gene deletion by using plaque purification technology⁺:BAC⁺See fig. 1;

(3) extracting infectious genome of the recombinant virus, co-transfecting cells with transfer plasmids pUC19: H1: H2, and screening the traceless recombinant virus rPRV FJ-2012-delta gE/gI without EGFP and BAC sequences by a plaque purification technology, wherein the genes of the traceless recombinant virus rPRV FJ-2012-delta gE/gI do not contain EGFP and BAC sequences, and the infectious genome of the recombinant virus is shown in figure 2.

The sequences of the upstream and downstream homologous fragments H1 and H2 are shown as SEQ ID NO.1-2, the sequence of the EGFP expression cassette is shown as SEQ ID NO.3, and the sequence of BAC is shown as SEQ ID NO. 4.

The PRV gE/gI gene deletion and deletion strain identification primer sequences and enzyme cutting sites thereof are shown in table 1, and the EGFP expression cassette amplification and EGFP and BAC identification primer sequences and enzyme cutting sites thereof are shown in table 2.

TABLE 1 identification primer sequences for PRV gE/gI gene deletion and deletion strains and restriction enzyme sites thereof

TABLE 2 EGFP expression cassette amplification and EGFP and BAC identification primer sequences and restriction enzyme sites thereof

A method for preparing a pseudorabies virus FJ-2012 strain gE/gI gene deletion strain inactivated vaccine by using the gene deletion strain comprises the following steps:

(1) propagating the virus; (2) inactivating the antigen; (3) preparing the inactivated vaccine.

The inactivated vaccine comprises a water-phase adjuvant vaccine and an oil adjuvant vaccine.

Further, the inactivated vaccine of the pseudorabies virus FJ-2012 strain gE/gI gene deletion strain is prepared by the method.

The invention has the advantages that:

according to the invention, a gE/gI gene deletion strain of a new pseudorabies virus variation FJ-2012 strain is used as a vaccine seed virus, formaldehyde is inactivated through amplification culture and then the inactivated vaccine is prepared into an inactivated vaccine immune sheep with different adjuvants, the inactivated vaccine is attacked by a parental virulent strain FJ-2012 strain after being immunized for 4 weeks, the inactivated vaccine provides complete immune protection effect on the immune sheep, and the method is suitable for identifying and diagnosing wild virus infection or vaccine immunity by using a general gE ELISA identification and diagnosis kit at present.

Drawings

FIG. 1 pUC 19H 1 EGFP BAC H2 and PRV FJ-2012 strains infectionrPRV with EGFP sequence formed after sexual DNA cotransfection of BHK-21 cells⁺:BAC⁺Recombinant virus, left panel under fluorescence, right panel under bright field virus cytopathic effect.

FIG. 2, pUC19, H1, H2 and rFJ-2012- Δ gE/gI EGFP, rPRV FJ-2012- Δ gE/gI recombinant virus of BAC sequence formed after co-transfection of BHK-21 cells with infectious DNA of BAC strain, left panel is virus cytopathic under bright field, right panel is recombinant virus under fluorescent field (showing no fluorescence).

Detailed Description

Example 1 preparation of inactivated vaccine for gE/I gene-deleted strain of Pseudorabies virus FJ-2012 Strain

1.1 Virus propagation and inactivation

And (3) virus propagation: adopting a standing culture mode to carry out virus mass propagation on a pseudorabies virus FJ-2012 strain gE/I gene deletion strain (named as a pseudorabies virus FJ-2012 delta gE/gI strain which is preserved in China center for type culture Collection in 3.8.2020 and has the preservation number of CCTCC NO: V202025), adopting BHK-21 cells in a square bottle, carrying out freeze thawing for 3 times after propagation, carrying out high-speed centrifugation at 15000rpm for 30min at 4 ℃ to remove cell debris, collecting supernatant as a pre-inactivation antigen for later use, and measuring TCID on the BHK-21 cells after 10 times of serial dilution₅₀The virus stock solution is serially diluted by 10 times by using serum-free DMED culture medium, and 10 times of the virus stock solution is put into a 96-well plate full of single-layer BHK-21 cells^-4、10^-5、10^-6、10^-7、10^-8、10^-9、10^-10Respectively inoculating 0.1 ml/well of diluted virus solution into cells (after culture solution is discarded), inoculating 8-well cells into each dilution, continuously culturing for 7 days, continuously observing whether each well of cells has typical cytopathic effect after PRV infection, and calculating TCID of virus according to Reed-Muench method₅₀. TCID of FJ-2012. delta. gE/gI strain was determined₅₀Are respectively 10^7.8/ml。

Antigen inactivation: slowly adding appropriate amount of 10% formaldehyde into the antigen virus solution to make the final concentration of formaldehyde be 0.1%, shaking, transferring into a new sterile triangular flask, inactivating at 37 deg.C for 36 hr, and continuously shaking during inactivationThe inactivation was completed by shaking at 200 rpm. After the inactivation, 5ml of inactivated virus solution was inoculated to 5 bottles of full monolayer BHK-21 cells (25 cm)²) 1ml of the culture solution is cultured for 7 days at 37 ℃ in each bottle, and whether cytopathic effect appears or not is observed to determine whether the virus is completely inactivated or not. The virus is completely inactivated by inspection.

1.2 preparation of inactivated vaccine

The virus liquid which is completely detected and inactivated is prepared by using a water-phase adjuvant GEL02 produced by Satipidae company and a self-made white oil adjuvant according to a using method, the prepared virus liquid is subpackaged after being prepared, the appearance and the quality of the subpackaged vaccine are detected, the requirements are met, the general process of the production process of the vaccine and the trial production work of laboratory products are basically completed in a laboratory, and the research on the effectiveness and the safety of the inactivated vaccine is planned to be developed.

1.2.1 preparation of aqueous adjuvant (GEL 02) vaccine

Preparing appropriate amount of adjuvant GEL02 (75 ml) and inactivated water phase antigen (425 ml), placing beaker containing water phase antigen under stirrer, stirring at 200rpm, adding adjuvant phase into water phase antigen, stirring at 200rpm for 10min, standing at 20 deg.C overnight, checking by QC, placing vaccine in glass bottle, storing at 3 temperatures (4 deg.C, 20 deg.C, 37 deg.C), and observing stability. 3 key points are required to be ensured during vaccine preparation, namely low shearing, sufficient stirring time and uniform emulsion are ensured respectively.

1.2.2 preparation of oil-adjuvanted (white oil) vaccines

Preparing an oil phase: adding 60ml of span into 600 ml of white oil, and fully stirring at 60 ℃; filtering with 0.2 μm PTFE filter membrane or autoclaving at 115 deg.C for 15 min.

Preparation of an aqueous phase: slowly adding 5ml of Tween-80 into 160ml of inactivated virus liquid, and stirring to fully dissolve and uniformly mix the Tween-80.

Emulsification: transferring the oil phase beaker containing 368ml to an emulsifying machine, adjusting the rotating speed of the emulsifying machine to 11000 rpm, mixing the oil phase for 15 s, gradually adding the water phase antigen into the oil phase, and emulsifying by the emulsifying machine at the temperature of less than 25 ℃. Checking that the prepared emulsion is milky emulsion, taking a clean suction pipe, sucking a small amount of vaccine and dripping the vaccine into cold water in an oil drop shape without diffusion; taking 3-5ml of emulsified sample, centrifuging at 1570g for 15min at 20 ℃, and no obvious layering. The emulsion is not broken after being placed for 21 days at the temperature of about 37 ℃.

Example 2 effectiveness test of PRV FJ-2012 Δ gE/gI Strain inactivated vaccine

2.1 test animals and grouping and handling

20 Hu sheep of 15-20kg are divided into 4 groups, and each group has 5 heads. The sheep is immunized by using PRV FJ-2012 delta gE/gI strain inactivated vaccines prepared by using GEL02 adjuvant and white oil adjuvant in a manner that 5ml of neck subcutaneous immune vaccines are used, 4 weeks later after immunization, the PRV FJ-2012 virulent strains are used for challenge, the dose is 1 ml/head, and 10 viruses are contained⁴ TCID₅₀. The groups of the test animals and the immunity and the toxicity attack are shown in the table 1. After challenge, body temperature was measured twice daily, and the mental state and disease incidence of animals were observed, including whether gnawing the inoculated part and whether neurological symptoms appeared, 14 days after challenge, the surviving animals were dissected and pathological changes were observed.

TABLE 1 grouping and treatment of test animals

2.2 results

The fourth day after the virulent strain FJ-2012 strain attacks the poison, 5 sheep in the G3 group had watery lying water samples, had bitten hind limbs, had white foam in the mouth, had depressed spirit and had a preference for lying, and 2 sheep died on the same day. No obvious clinical symptoms appeared in groups G1, G2, G4.

On the fifth day after challenge, 3 sheep in the G3 group all showed watery limbs, no stooping, and white foam in the mouth, and all 3 sheep died on the same day. No obvious clinical symptoms appeared in groups G1, G3, G4.

On the sixth to fourteen days after the challenge, no obvious clinical symptoms appeared in groups G1, G3, and G4. Specific morbidity and mortality results are shown in table 2.

TABLE 2 protection rates of different adjuvant vaccine immunizations against challenge with PRV FJ-2012 virulent strain

The immune sera collected before and 4 weeks after immunization were tested by ELISA and all the antibodies gB and gE of groups G1, G2, G3 and G4 before immunization were negative, indicating no PRV infection. ELISA results after 4 weeks of immunization show that all gB antibodies of G1 and G2 group sheep are positive (Table 3), and all gE antibodies are negative (Table 3), so that the inoculation effect of the FJ-2012 delta gE/gI strain inactivated vaccine prepared by two different adjuvants is good, and the kit can be suitable for the current universal commercial gE differential diagnosis ELISA kit.

TABLE 3 ELISA determination of serum gB and gE antibody results after 28 d immunization of sheep with PRV FJ-2012 Δ gE/gI strain inactivated vaccine with different adjuvants

^*PRV gB ELISA S/N＜0.60 = Positive; PRV gB ELISA S/N＞0.60 and≤0.70 = Suspect; PRV gB ELISA S/N＞0.70 = Negative.

§PRV gE ELISA S/N＜0.60 = Positive; PRV gE ELISA S/N＞0.60 and≤0.70 = Suspect; PRV gE ELISA S/N＞0.70 = Negative.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province

<120> preparation and application of inactivated vaccine for gE/gI gene deletion strain of pseudorabies virus FJ-2012 strain

<130> 15

<160> 15

<170> PatentIn version 3.3

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gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc 1200

catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata 1260

aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt 1320

ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca 1380

ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc 1440

tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg 1500

aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg 1560

cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac 1620

aggtatttat tcgcgataag ctcatggagc ggcgtaaccg tcgcacagga aggacagaga 1680

aagcgcggat ctgggaagtg acggacagaa cggtcaggac ctggattggg gaggcggttg 1740

ccgccgctgc tgctgacggt gtgacgttct ctgttccggt cacaccacat acgttccgcc 1800

attcctatgc gatgcacatg ctgtatgccg gtataccgct gaaagttctg caaagcctga 1860

tgggacataa gtccatcagt tcaacggaag tctacacgaa ggtttttgcg ctggatgtgg 1920

ctgcccggca ccgggtgcag tttgcgatgc cggagtctga tgcggttgcg atgctgaaac 1980

aattatcctg agaataaatg ccttggcctt tatatggaaa tgtggaactg agtggatatg 2040

ctgtttttgt ctgttaaaca gagaagctgg ctgttatcca ctgagaagcg aacgaaacag 2100

tcgggaaaat ctcccattat cgtagagatc cgcattatta atctcaggag cctgtgtagc 2160

gtttatagga agtagtgttc tgtcatgatg cctgcaagcg gtaacgaaaa cgatttgaat 2220

atgccttcag gaacaataga aatcttcgtg cggtgttacg ttgaagtgga gcggattatg 2280

tcagcaatgg acagaacaac ctaatgaaca cagaaccatg atgtggtctg tccttttaca 2340

gccagtagtg ctcgccgcag tcgagcgaca gggcgaagcc ctcgagtgag cgaggaagca 2400

ccagggaaca gcacttatat attctgctta cacacgatgc ctgaaaaaac ttcccttggg 2460

gttatccact tatccacggg gatattttta taattatttt ttttatagtt tttagatctt 2520

cttttttaga gcgccttgta ggcctttatc catgctggtt ctagagaagg tgttgtgaca 2580

aattgccctt tcagtgtgac aaatcaccct caaatgacag tcctgtctgt gacaaattgc 2640

ccttaaccct gtgacaaatt gccctcagaa gaagctgttt tttcacaaag ttatccctgc 2700

ttattgactc ttttttattt agtgtgacaa tctaaaaact tgtcacactt cacatggatc 2760

tgtcatggcg gaaacagcgg ttatcaatca caagaaacgt aaaaatagcc cgcgaatcgt 2820

ccagtcaaac gacctcactg aggcggcata tagtctctcc cgggatcaaa aacgtatgct 2880

gtatctgttc gttgaccaga tcagaaaatc tgatggcacc ctacaggaac atgacggtat 2940

ctgcgagatc catgttgcta aatatgctga aatattcgga ttgacctctg cggaagccag 3000

taaggatata cggcaggcat tgaagagttt cgcggggaag gaagtggttt tttatcgccc 3060

tgaagaggat gccggcgatg aaaaaggcta tgaatctttt ccttggttta tcaaacgtgc 3120

gcacagtcca tccagagggc tttacagtgt acatatcaac ccatatctca ttcccttctt 3180

tatcgggtta cagaaccggt ttacgcagtt tcggcttagt gaaacaaaag aaatcaccaa 3240

tccgtatgcc atgcgtttat acgaatccct gtgtcagtat cgtaagccgg atggctcagg 3300

catcgtctct ctgaaaatcg actggatcat agagcgttac cagctgcctc aaagttacca 3360

gcgtatgcct gacttccgcc gccgcttcct gcaggtctgt gttaatgaga tcaacagcag 3420

aactccaatg cgcctctcat acattgagaa aaagaaaggc cgccagacga ctcatatcgt 3480

attttccttc cgcgatatca cttccatgac gacaggatag tctgagggtt atctgtcaca 3540

gatttgaggg tggttcgtca catttgttct gacctactga gggtaatttg tcacagtttt 3600

gctgtttcct tcagcctgca tggattttct catacttttt gaactgtaat ttttaaggaa 3660

gccaaatttg agggcagttt gtcacagttg atttccttct ctttcccttc gtcatgtgac 3720

ctgatatcgg gggttagttc gtcatcattg atgagggttg attatcacag tttattactc 3780

tgaattggct atccgcgtgt gtacctctac ctggagtttt tcccacggtg gatatttctt 3840

cttgcgctga gcgtaagagc tatctgacag aacagttctt ctttgcttcc tcgccagttc 3900

gctcgctatg ctcggttaca cggctgcggc gagcgctagt gataataagt gactgaggta 3960

tgtgctcttc ttatctcctt ttgtagtgtt gctcttattt taaacaactt tgcggttttt 4020

tgatgacttt gcgattttgt tgttgctttg cagtaaattg caagatttaa taaaaaaacg 4080

caaagcaatg attaaaggat gttcagaatg aaactcatgg aaacacttaa ccagtgcata 4140

aacgctggtc atgaaatgac gaaggctatc gccattgcac agtttaatga tgacagcccg 4200

gaagcgagga aaataacccg gcgctggaga ataggtgaag cagcggattt agttggggtt 4260

tcttctcagg ctatcagaga tgccgagaaa gcagggcgac taccgcaccc ggatatggaa 4320

attcgaggac gggttgagca acgtgttggt tatacaattg aacaaattaa tcatatgcgt 4380

gatgtgtttg gtacgcgatt gcgacgtgct gaagacgtat ttccaccggt gatcggggtt 4440

gctgcccata aaggtggcgt ttacaaaacc tcagtttctg ttcatcttgc tcaggatctg 4500

gctctgaagg ggctacgtgt tttgctcgtg gaaggtaacg acccccaggg aacagcctca 4560

atgtatcacg gatgggtacc agatcttcat attcatgcag aagacactct cctgcctttc 4620

tatcttgggg aaaaggacga tgtcacttat gcaataaagc ccacttgctg gccggggctt 4680

gacattattc cttcctgtct ggctctgcac cgtattgaaa ctgagttaat gggcaaattt 4740

gatgaaggta aactgcccac cgatccacac ctgatgctcc gactggccat tgaaactgtt 4800

gctcatgact atgatgtcat agttattgac agcgcgccta acctgggtat cggcacgatt 4860

aatgtcgtat gtgctgctga tgtgctgatt gttcccacgc ctgctgagtt gtttgactac 4920

acctccgcac tgcagttttt cgatatgctt cgtgatctgc tcaagaacgt tgatcttaaa 4980

gggttcgagc ctgatgtacg tattttgctt accaaataca gcaatagtaa tggctctcag 5040

tccccgtgga tggaggagca aattcgggat gcctggggaa gcatggttct aaaaaatgtt 5100

gtacgtgaaa cggatgaagt tggtaaaggt cagatccgga tgagaactgt ttttgaacag 5160

gccattgatc aacgctcttc aactggtgcc tggagaaatg ctctttctat ttgggaacct 5220

gtctgcaatg aaattttcga tcgtctgatt aaaccacgct gggagattag ataatgaagc 5280

gtgcgcctgt tattccaaaa catacgctca atactcaacc ggttgaagat acttcgttat 5340

cgacaccagc tgccccgatg gtggattcgt taattgcgcg cgtaggagta atggctcgcg 5400

gtaatgccat tactttgcct gtatgtggtc gggatgtgaa gtttactctt gaagtgctcc 5460

ggggtgatag tgttgagaag acctctcggg tatggtcagg taatgaacgt gaccaggagc 5520

tgcttactga ggacgcactg gatgatctca tcccttcttt tctactgact ggtcaacaga 5580

caccggcgtt cggtcgaaga gtatctggtg tcatagaaat tgccgatggg agtcgccgtc 5640

gtaaagctgc tgcacttacc gaaagtgatt atcgtgttct ggttggcgag ctggatgatg 5700

agcagatggc tgcattatcc agattgggta acgattatcg cccaacaagt gcttatgaac 5760

gtggtcagcg ttatgcaagc cgattgcaga atgaatttgc tggaaatatt tctgcgctgg 5820

ctgatgcgga aaatatttca cgtaagatta ttacccgctg tatcaacacc gccaaattgc 5880

ctaaatcagt tgttgctctt ttttctcacc ccggtgaact atctgcccgg tcaggtgatg 5940

cacttcaaaa agcctttaca gataaagagg aattacttaa gcagcaggca tctaaccttc 6000

atgagcagaa aaaagctggg gtgatatttg aagctgaaga agttatcact cttttaactt 6060

ctgtgcttaa aacgtcatct gcatcaagaa ctagtttaag ctcacgacat cagtttgctc 6120

ctggagcgac agtattgtat aagggcgata aaatggtgct taacctggac aggtctcgtg 6180

ttccaactga gtgtatagag aaaattgagg ccattcttaa ggaacttgaa aagccagcac 6240

cctgatgcga ccacgtttta gtctacgttt atctgtcttt acttaatgtc ctttgttaca 6300

ggccagaaag cataactggc ctgaatattc tctctgggcc cactgttcca cttgtatcgt 6360

cggtctgata atcagactgg gaccacggtc ccactcgtat cgtcggtctg attattagtc 6420

tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac 6480

tcgtatcgtc ggtctgataa tcagactggg accacggtcc cactcgtatc gtcggtctga 6540

ttattagtct gggaccatgg tcccactcgt atcgtcggtc tgattattag tctgggacca 6600

cggtcccact cgtatcgtcg gtctgattat tagtctggaa ccacggtccc actcgtatcg 6660

tcggtctgat tattagtctg ggaccacggt cccactcgta tcgtcggtct gattattagt 6720

ctgggaccac gatcccactc gtgttgtcgg tctgattatc ggtctgggac cacggtccca 6780

cttgtattgt cgatcagact atcagcgtga gactacgatt ccatcaatgc ctgtcaaggg 6840

caagtattga catgtcgtcg taacctgtag aacggagtaa cctcggtgtg cggttgtatg 6900

cctgctgtgg attgctgctg tgtcctgctt atccacaaca ttttgcgcac ggttatgtgg 6960

acaaaatacc tggttaccca ggccgtgccg gcacgttaac cgggctgcat ccgatgcaag 7020

tgtgtcgctg tcgacgagct cgcgagctcg gacatgaggt tgccccgtat tcagtgtcgc 7080

tgatttgtat tgtctgaagt tgtttttacg ttaagttgat gcagatcaat taatacgata 7140

cctgcgtcat aattgattat ttgacgtggt ttgatggcct ccacgcacgt tgtgatatgt 7200

agatgataat cattatcact ttacgggtcc tttccggtga tccgacaggt tacggggcgg 7260

cgacctcgcg ggttttcgct atttatgaaa attttccggt ttaaggcgtt tccgttcttc 7320

ttcgtcataa cttaatgttt ttatttaaaa taccctctga aaagaaagga aacgacaggt 7380

gctgaaagcg agctttttgg cctctgtcgt ttcctttctc tgtttttgtc cgtggaatga 7440

acaatggaag tccgagctca tcgctaataa cttcgtatag catacattat acgaagttat 7500

attcgat 7507

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence

<400> 5

attgaattcg tacccgtaca ccgagtcgt 29

<210> 6

<211> 39

<212> DNA

<213> Artificial sequence

<400> 6

actgagctcg gttaattaat catcatcgac gccggtact 39

<210> 7

<211> 39

<212> DNA

<213> Artificial sequence

<400> 7

caactgcagc gttaattaag ccgacatgga cacgttcga 39

<210> 8

<211> 27

<212> DNA

<213> Artificial sequence

<400> 8

tcgaagcttt tgtggacccg cgaacat 27

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence

<400> 9

tccactcgca gctcttct 18

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence

<400> 10

gcacgtcatc acgaagga 18

<210> 11

<211> 41

<212> DNA

<213> Artificial sequence

<400> 11

gcgttaatta agcatgctag ttattaatag taatcaatta c 41

<210> 12

<211> 34

<212> DNA

<213> Artificial sequence

<400> 12

gacttaatta aagatacatt gatgagtttg gaca 34

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<400> 13

aggacgacgg caactacaag 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

tctcgttggg gtctttgctc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<400> 15

atctggctct gaaggggcta 20

Claims (6)

1. A pseudorabies virus FJ-2012 strain gE/gI gene deletion strain is characterized in that the gene deletion strain is named as a pseudorabies virus FJ-2012 delta gE/gI strain, is preserved in China center for type culture collection (CCTCC NO: V202025) at 8/4/2020, and has a preservation number of CCTCC NO: V202025.

2. The method for constructing the pseudorabies virus FJ-2012 strain gE/I gene deletion strain according to claim 1, which comprises the following steps:

(1) sequentially inserting upstream and downstream homologous fragments of a gE/I deletion position of an FJ-2012 strain into a plasmid pUC19 to construct pUC19: H1: H2, then inserting an EGFP expression cassette to construct a homologous recombination transfer plasmid pUC19: H1: EGFP: H2, and then introducing a BAC sequence into pUC19: H1: EGFP: H2 to form a homologous recombination transfer plasmid pUC19: H1: EGFP: BAC 2 capable of constructing a PRV artificial chromosome;

(2) extracting infectious genome DNA of PRV FJ-2012 strain, co-transfecting cells with transfer plasmids pUC19, H1, EGFP, BAC, H2, and screening recombinant virus rPRV FJ-2012-delta gE/gI and EGFP containing fluorescent marker gE/I gene deletion by using plaque purification technology⁺:BAC⁺；

(3) Extraction and recombinationEGFP, a virus rPRV FJ-2012- Δ gE/gI⁺:BAC⁺Co-transfecting cells with transfer plasmids pUC19: H1: H2, and screening the traceless recombinant virus rPRV FJ-2012-delta gE/gI without EGFP and BAC sequences with gE/I gene deletion by a plaque purification technology.

3. The construction method of claim 2, wherein the sequences of the upstream and downstream homologous fragments H1 and H2 are shown as SEQ ID NO.1-2, the sequence of the EGFP expression cassette is shown as SEQ ID NO.3, and the sequence of the BAC is shown as SEQ ID NO. 4.

4. A method for preparing inactivated vaccine of the pseudorabies virus FJ-2012 strain gE/gI gene deletion strain by using the gene deletion strain in claim 1, which is characterized by comprising the following steps:

(1) propagating the virus; (2) inactivating the antigen; (3) preparing the inactivated vaccine.

5. The method of claim 2, wherein the inactivated vaccine comprises an aqueous-phase adjuvant vaccine, an oil-adjuvant vaccine.

6. The inactivated vaccine of the gE/gI gene deletion strain of the pseudorabies virus FJ-2012 strain prepared by the method of claim 2.

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