CN112626038B

CN112626038B - Construction of pseudorabies virus FB strain gE/gI gene deletion strain and application thereof as vaccine

Info

Publication number: CN112626038B
Application number: CN202110152612.2A
Authority: CN
Inventors: 周伦江; 侯博; 王晨燕; 刘玉涛; 王隆柏; 陈秋勇; 吴学敏; 陈少莺
Original assignee: Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Current assignee: Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2023-01-10
Anticipated expiration: 2041-02-04
Also published as: CN112626038A

Abstract

According to the invention, the gE/gI gene of the PRV artificial attenuated strain FB is deleted by adopting a gene homologous recombination method, the gE/gI gene has a gene marker for identifying and diagnosing wild virus infection, and is suitable for a general gE ELISA identifying and diagnosing kit at present, and an inactivated vaccine prepared by taking the deleted strain as a seed virus can generate a complete immune protection effect on the attack of a PRV new mutation super-virulent strain FJ-2012; compared with Bartha-K61 vaccine strain, the FB gE/gI gene deletion strain has higher safety, so the FB gE/gI gene deletion strain is also suitable to be used as a weak-virus live vaccine candidate strain for preventing the infection of the new mutation PRV super-virulent strain.

Description

Construction of pseudorabies virus FB strain gE/gI gene deletion strain and application thereof as vaccine

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to construction of a pseudorabies virus FB strain gE/gI gene deletion strain and application of the pseudorabies virus FB strain as a 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). PRV infection many mammals, including pigs, cattle, sheep, goats, dogs, rabbits, boars, mink, bears, cats, foxes, mice and wild mice, cause severe neurological symptoms and death in addition to pigs. Pigs are the only viable animal after PRV infection and therefore the storage host for the virus and the only repository for PRV. The disease brings huge economic loss to the pig breeding industry all over the world, but part of European and American countries invest huge manpower, material resources and financial resources for the disease, and the disease is purified and eliminated in domestic pigs through 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 10 months in 2011, 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 to fattening pigs of 85 days, so that serious economic loss is caused to the pig industry of China, and the method is mainly characterized in that: (i) Pregnant sows immunized with the Bartha-K61 vaccine develop high miscarriage rates; (ii) Growing pigs develop central nervous system disorders and have high mortality; (iii) high GE-positivity of serum.

PRV causes great trouble to the pig farming industry of countries in the world, particularly countries with large pig production, including south America, europe and Asia, but through the large-scale use of gE-deficient vaccines and DIVA (differentiated fed from preserved animals, DIVA) differential diagnosis technology, the U.S. and some European countries have eradicated porcine pseudorabies from domestic pigs, but PRV is still circulating and spreading in wild herds in the U.S., germany and other countries. Pseudorabies cases were first reported in 1950s in China, and in 1970s, bartha-K61 vaccine strains were introduced into China, and PR was well controlled from 1990s to 2011 using Bartha-K61 vaccine strains or local gE-deleted attenuated vaccines. However, since 10 months 2011, the large-scale outbreak of pseudorabies unprecedentedly produced by large-scale pig farms immunized with Bartha-K61 vaccine in China and spread to other provinces and cities in China rapidly due to the fact that the pseudorabies is rolled up nationwide, so that the pseudorabies is devastating to the pig industry in China. Research shows that compared with the classical PRV strains, the PRV strains separated from large-scale pig farms in different popular areas all show increased toxicity, increase the pathogenicity to fattening pigs and piglets, and can cause death of big pigs in the fattening pigs, and the existing vaccine can not provide good protection for the newly-generated variant strains and is the newly-generated variant PRV super-virulent strain. All newly-developed variant ultrastrong PRV strains isolated after 2011 have significant variations 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 are separated from different areas of China, including HNX, ZJ01, heN1, SMX, TJ, JS, HNB, HN1201, FJ-2012 and the like. Therefore, the PRV variation and virulence enhancement in China bring serious troubles to the pig industry at the present stage of China.

In conclusion, there is a need for a safe and effective vaccine for the prevention and control of classical PRV and new variant PRV infections, and for differential diagnosis. According to the invention, the gE/gI gene of the PRV artificial attenuated strain FB is deleted by adopting a gene homologous recombination method, the gE/gI gene has a gene marker for identifying and diagnosing wild virus infection, and is suitable for a general gE ELISA identifying and diagnosing kit at present, and an inactivated vaccine prepared by taking the deleted strain as a seed virus can generate a complete immune protection effect on the attack of a PRV new mutation super-virulent strain FJ-2012; compared with Bartha-K61 vaccine strain, the FB gE/gI gene deletion strain has higher safety, so the FB gE/gI gene deletion strain is also suitable to be used as a weak-virus live vaccine candidate strain for preventing the infection of the new mutation PRV super-virulent strain.

Disclosure of Invention

The invention aims to provide a construction of a pseudorabies virus FB strain gE/gI gene deletion strain and application thereof as a vaccine.

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

a construction method of a gE/gI gene deletion strain of a pseudorabies virus FB strain comprises the following steps:

(1) Sequentially inserting upstream and downstream homologous fragments of a gE/I deletion position of an FB strain into a plasmid pUC19 to construct pUC19: H1: H2, then inserting an EGFP expression cassette, constructing 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 FB strain, cotransfecting the PRV FB strain and transfer plasmid pUC19: H1: EGFP: BAC: H2 into cells, and screening recombinant virus rPRV FB-delta gE/gI: EGFP containing fluorescent marker gE/I gene deletion by a plaque purification technology ⁺ :BAC ⁺ ；

(3) Extracting rPRV FB-delta gE/gI EGFP ⁺ :BAC ⁺ The infectious genome is transfected with a transfer plasmid pUC19: H1: H2 together to obtain a traceless recombinant virus rPRV FB-delta gE/gI with gE/I gene deletion and without EGFP and BAC sequences by a plaque purification technology.

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 BAC sequence is shown as SEQ ID NO. 4.

The construction method of the recombinant virus in the step (2) comprises the following steps:

(1) Extraction of circular PRV genomic DNA:

1) Inoculating PRV virus to full monolayer BHK-21 cells, digesting with pancreatin when 30% cytopathic effect appears, centrifuging, and collecting cells;

2) Resuspending the collected cells with 400 μ L PBS, adding lysate, mixing well, and digesting overnight;

3) Equal volume of phenol was added: chloroform: mixing the isoamyl alcohol mixed solution evenly, and centrifuging at 12000rpm for 10min at 4 ℃;

4) The supernatant was taken and added again with an equal volume of phenol: chloroform: mixing the isoamyl alcohol mixed solution evenly, and centrifuging at 12000rpm for 10min at 4 ℃;

5) Repeating the step 4) once;

6) Transferring the supernatant into a new centrifuge tube, adding 1/10 volume of 3M sodium acetate and 2 times volume of absolute ethyl alcohol, mixing uniformly, and precipitating at-20 ℃ overnight;

7) Centrifuging at 12000rpm at 4 deg.C for 10min, discarding supernatant, washing precipitate with precooled 75% ethanol, centrifuging at 7600rpm for 5min, discarding supernatant, air drying, adding 30 μ L ddH ₂ O dissolving DNA for later use;

(2) EGFP as recombinant virus rPRV FB-delta gE/gI ⁺ :BAC ⁺ Constructing, purifying and identifying; washing BHK-21 cells growing to 80% in a 6-well plate with opti-MEM culture medium for 2 times, adding all transfection solution into the washed cells, after 6 hours of transfection, changing DMEM culture medium containing 10% FBS, continuously culturing for 48-72 hours, collecting cells and culture solution thereof according to cell morphology, after 3 times of freeze-thawing, inoculating all collected virus solution into 1-well BHK-21 cells in a 6-well plate, and after 24-48 hours of culture, observing whether fluorescence is generated and cytopathic effect is generated under a fluorescence microscope;

the transfection solution formula comprises: 2mL of opti-MEM medium; lipofectamine 2000 μ L; 10. Mu.L of PRV DNA; pUC19: H1: EGFP: BAC: H2. Mu.L.

Further, the pseudorabies virus FB strain gE/gI gene deletion strain is prepared by the method, is named as a pseudorabies virus FB delta gE/gI strain, is preserved in China center for type culture Collection at 8.4.2020, and has the preservation number of CCTCC NO: V202026. The China center for type culture Collection is addressed to Wuhan, wuhan university, china.

Further, the deletion strain is applied to the preparation of a gE/I gene deletion inactivated vaccine of the pseudorabies virus FB strain, including the preparation of a biphasic adjuvant inactivated vaccine.

The invention has the advantages that:

according to the invention, the gE/gI gene of the PRV artificial attenuated strain FB is deleted by adopting a gene homologous recombination method, so that the gE/gI gene has a gene marker for identifying and diagnosing wild virus infection, and is suitable for a general gE ELISA identification and diagnosis kit at present, and an inactivated vaccine prepared by taking the deleted strain as a seed virus can generate a complete immune protection effect on the attack of a PRV new mutation hyper-virulent strain FJ-2012; compared with Bartha-K61 vaccine strain, the FB gE/gI gene deletion strain has higher safety, so the FB gE/gI gene deletion strain is also suitable to be used as a weak-virus live vaccine candidate strain for preventing the infection of the new mutation PRV super-virulent strain.

Drawings

FIG. 1 shows the PCR amplification results of homologous arm fragments H1 and H2 at upstream and downstream of gE/gI gene of PRV FB strain;

FIG. 2 shows the results of double restriction enzyme digestion of plasmid pUC19: H1: H2;

FIG. 3 shows the results of PCR identification of the EGPF and BAC sequences of the pUC19: H1: EGFP: BAC: H2 plasmids;

FIG. 4 shows that rPRV FB-delta gE/gI with EGFP sequence formed after infectious DNA of pUC19: H1: EGFP: BAC: H2 and PRV FB strain co-transfect BHK-21 cells ⁺ :BAC ⁺ Recombinant virus, left picture is recombinant virus under fluorescence, right picture is virus cytopathic effect under bright field;

FIG. 5 shows recombinant viruses deleted EGFP of BAC sequence rPRV FB- Δ gE/gI, formed after co-transfection of BHK-21 cells with pUC19: H1: H2 and rFB- Δ gE/gI EGFP strain infectious DNA, the left picture shows virus cytopathy in bright field, the right picture shows recombinant viruses in fluorescent field (showing no fluorescence);

FIG. 6 shows clinical symptoms in sheep; a) The symptoms of itching. B) The itching area had hair removed and was red and swollen. C) White foam is spitted out from the mouth.

Detailed Description

Example 1 construction of a Strain lacking gE/I Gene of Pseudorabies Virus FB Strain

A method for preparing pseudorabies virus FB strain is disclosed in Wei Zhenming, a section of bolt, li Yiying, et al, research on the culture of pseudorabies virus attenuated strains [ J ]. Chinese veterinary science and technology, 1986 (08): 7-9; li Yiying, wei Zhenming, wu Ping, et al, several biological characteristics of pseudorabies virus attenuated strains [ J ]. Proc. Nongc academy of agricultural sciences, 1995, 10 (04): 20-25; wei Zhenming, by latchless, weng Wenlin, et al, pseudorabies attenuated vaccine research-observation of passive immunization effect of piglets [ J ]. Chinese veterinary science, 1989 (05): 7-9.

1.1 Construction of homologous recombination transfer plasmids pUC19: H1: EGFP: BAC: H2

By using FB strain DNA as a template, sequentially inserting upstream and downstream homologous fragments of a gE/I deletion position 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 the homologous recombination transfer plasmid pUC19: H1: EGFP: BAC 2 capable of constructing a PRV artificial chromosome.

1.1.1 Construction of transfer plasmid pUC19: H1: H2

Primers were designed (see Table 1) to amplify the upstream fragment H1 and the downstream fragment H2 of the homologous recombination arm, respectively, in view of the primer sequences of PRV gE/gI gene deletions in Ph.RTM.Sam.2014.

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

The reaction system for PCR amplification of H1 and H2 is as follows:

the reaction procedure was as follows: 95. 5min at the temperature; 95. 35 cycles of 30s, 30s at 60 ℃ and 70 ℃ at 68 ℃ and finally an extension at 68 ℃ for 10 min.

Separating PCR products of an upstream and downstream sequence H1 fragment and an H2 fragment of PRV gE/gI gene by agarose gel electrophoresis, preliminarily confirming that the amplification result is correct (figure 1), recovering and purifying according to the specification of a gel cutting recovery kit of Tiangen company, respectively connecting and cloning the H1 and H2 fragments into a blunt-end cloning vector pCR ^® -Blunt II-TOPO ^® And (3) a carrier. The kit was purchased from thermo Fisher company under the accession number K280002 and the linkage system was as follows:

connecting the reaction system at 22.5 ℃ for 30min, adding the connection product into DH5 alpha escherichia coli competent cells, carrying out ice bath for 30min, carrying out water bath for 90s at 42 ℃, carrying out ice bath for 90s, then adding 250 mu L of non-resistant LB culture medium, incubating at 37 ℃ for 45-60min, taking 200 mu L of LB plate coated with 30 mu g/mL kanamycin, carrying out overnight culture at 37 ℃, selecting a single colony for culture, extracting plasmids by using a Qiagen miniprep kit, carrying out PCR identification, sending positive clones to a company for sequencing confirmation, selecting correctly sequenced plasmids pCR ^® -Blunt II-TOPO ^® H1 and pCR ^® -Blunt II-TOPO ^® H2 was subjected to subsequent tests.

Restriction enzymes for H1 fragment-containing plasmid and pUC19 plasmidEcoRI (NEB Corp.) andSaci (NEB) was subjected to double digestion,EcoRi, an enzyme digestion reaction system is as follows:

after digesting for 2 hours at 37 ℃, purifying by using a DNA column purification kit of Tiangen company, and then performingSacI, enzyme digestion, wherein the reaction system is as follows:

the cleavage products were separated by agarose gel electrophoresis, and then the target DNA was purified and recovered using a gel cutting recovery kit from Tiangen corporation, and after recovery, the H1 fragment and the pUC19 plasmid were ligated by T4 DNA ligase (NEB corporation) in the following manner:

connecting the reaction system at 22.5 ℃ for 1 hour, adding the connecting product into DH5 alpha escherichia coli competent cells, carrying out ice bath for 30min, carrying out water bath for 90s at 42 ℃ and carrying out ice bath for 90s, then adding 250 mu L of non-resistant LB culture medium, incubating at 37 ℃ for 45-60min, taking 200 mu LCoating LB plate containing 50 ug/mL ampicillin, culturing overnight at 37 deg.C, selecting single colony, culturing, extracting plasmid with Qiagen miniprep kit, and extracting with restriction enzymeEcoRI andHind III (NEB) performs double enzyme digestion identification, and the reaction system is as follows:

the reaction system is subjected to agarose gel electrophoresis detection after enzyme digestion for 2 hours at 37 ℃, and correctly identified plasmids are named as: pUC 19H 1.

pCR containing H2 fragment ^® -Blunt II-TOPO ^® Restriction enzymes for H2 plasmid and pUC19: H1 plasmidPstI (NEB Corp.) andHindIII (NEB) the reaction system is as follows:

after 3 hours of digestion at 37 ℃ and separation by agarose gel electrophoresis, the H2 fragment and the pUC19: H1 linearized target DNA were recovered by purification using a gel cutting recovery kit from Tiangen corporation, and the recovered H2 fragment and pUC19: H1 plasmid were ligated by T4 DNA ligase (NEB) in the following manner:

connecting the reaction system at 22.5 ℃ for 1 hour, adding the connection product into DH5 alpha escherichia coli competent cells, carrying out ice bath for 30min, carrying out water bath for 90s at 42 ℃, carrying out ice bath for 90s, then adding 250 mu L of non-resistant LB culture medium, incubating at 37 ℃ for 45-60min, taking 200 mu L of LB plate coated with 50 mu g/mL ampicillin, carrying out overnight culture at 37 ℃, selecting a single colony for culture, extracting plasmids by using a Qiagen miniprep kit, and using restriction enzyme for restriction enzyme digestionHind III andPsti, carrying out double enzyme digestion identification, wherein the fragment is large after enzyme digestionAbout 4000bp and 1100bp, the correctly identified plasmids were designated: pUC19: H1: H2 (see FIG. 2 for the cleavage results).

1.1.2 Construction of transfer plasmid pUC19: H1: EGFP: H2

The EGFP expression cassette is amplified by adopting the following primers and taking pEGFP-N1 as a template, and an amplification reaction system is as follows:

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

Remarking: EGFP-D-F and EGFP-D-F amplify EGFP sequences; BAC-D-F and BAC-D-R amplify the sopA gene of the BAC sequence.

The reaction procedure was as follows: 5min at 95 ℃; 32 cycles of 95 ℃ 30s,53 ℃ 30s,68 ℃ 2 min, and finally an extension of 10min at 68 ℃.

Separating PCR product by 1% agarose gel electrophoresis, primarily confirming that the amplification result is correct, recovering and purifying according to the specification of gel cutting recovery kit of Tiangen company, and using restriction enzyme to recover EGFP DNA fragment and pUC19: H1: H2 plasmidPacThe enzyme digestion was carried out according to the following reaction scheme:

after digesting at 37 ℃ for 15min, alkaline phosphatase (CIP) (NEB Co.) was added to the digested pUC19: H1: H2 plasmid in an amount of 1. Mu.L, 10 XBuffer 1. Mu.L, and dd H ₂ O8. Mu.L, total volume 60. Mu.L, and incubation was continued at 37 ℃ for 30min. Separating pUC19: H1: H2 treated by CIP and digested EGFP by agarose gel electrophoresis, and purifying the EGFP fragment and the linear pUC19: H1: H2 plasmid by a gel cutting recovery kit of Tiangen corporationThe ligation reaction system was as follows, in which the two were ligated with T4 DNA ligase (NEB) after recovery:

connecting the reaction system at 22.5 ℃ for 30min, adding the connection product into DH10B escherichia coli competent cells, carrying out ice bath for 30min, carrying out water bath for 90s at 42 ℃, carrying out ice bath for 90s, then adding 700 mu L of non-resistant LB culture medium, incubating at 37 ℃ for 45-60min, taking 200 mu L of LB plate coated with 50 mu g/mL ampicillin, carrying out overnight culture at 37 ℃, selecting a single colony for culture, carrying out colony PCR identification, wherein the identification primers are shown in Table 2, and the reaction system is as follows:

the reaction procedure was as follows: 5min at 95 ℃; 30 cycles of 95 ℃ 30s,58 ℃ 30s,72 ℃ 30s, and finally an extension at 72 ℃ for 10 min. The PCR product was detected by 1% agarose gel electrophoresis and the correctly identified plasmid was named: pUC19: H1: EGFP: H2 (PCR results are shown in FIG. 3A).

1.1.3 Construction of pUC19: H1: EGFP: BAC: H2 plasmids

Restriction enzymes were used for pUC19: H1: EGFP: H2 and pBelo BAC II plasmidsSph I (NEB) was digested as follows:

at 37 ℃, after enzyme digestion is carried out for 1 hour, the mixture is directly purified by a DNA column-passing purification kit of Tiangen corporation, two linearized plasmids after enzyme digestion are recovered, the two linearized plasmids are connected by T4 DNA ligase after recovery, and the connection reaction system is as follows:

connecting the reaction system at 20-21 ℃ for 2.5 hours, taking 10 mu L of the connection product, adding the connection product into DH5 alpha escherichia coli competent cells, carrying out ice bath for 30min, carrying out water bath for 90s at 42 ℃ and carrying out ice bath for 90s, then adding 250 mu L of nonresistant LB culture medium, incubating for 45-60min at 37 ℃, taking 200 mu L of LB plates coated with 50 mu g/mL ampicillin and 25 mu g/mL chloramphenicol, carrying out overnight culture at 37 ℃, selecting a single colony for culture, carrying out colony PCR identification, wherein the identification primers are shown in Table 2, and the reaction system is as follows:

the reaction procedure was as follows: 5min at 95 ℃; 30 cycles of 95 ℃ 30s,58 ℃ 30s,72 ℃ 30s, and finally an extension at 72 ℃ for 10 min. The PCR product was detected by 1% agarose gel electrophoresis and the correctly identified plasmid was named: pUC19: H1: EGFP: BAC: H2 (PCR results are shown in FIG. 3B).

1.2 Construction of recombinant virus rPRV FB-delta gE/gI EGFP ⁺ :BAC ⁺

Extracting genome infectious DNA of PRV FB strain, cotransfecting the PRV FB strain and transfer plasmid pUC19: H1: EGFP: BAC: H2 into cells, and screening recombinant virus rPRV FB-delta gE/gI: EGFP containing fluorescent marker gE/I gene deletion by a plaque purification technology ⁺ :BAC ⁺ 。

1.2.1 Extraction of circular PRV genomic DNA

1) Confluent monolayers of BHK-21 cells (25 cm) ² ) Inoculating PRV virus, digesting with pancreatin when 30% of cytopathic effect appears, centrifuging, and collecting cells;

5) Repeating the step 4) once;

7) Centrifuging at 12000rpm at 4 deg.C for 10min, discarding supernatant, washing precipitate with precooled 75% ethanol, centrifuging at 7600rpm for 5min, discarding supernatant, air drying, adding 30 μ L ddH ₂ O dissolving the DNA for later use.

1.2.2 EGFP as recombinant virus rPRV FB-delta gE/gI ⁺ :BAC ⁺ Construction, purification and characterization of

The transfection-verified pUC19: H1: EGFP: BAC: H2 plasmid and the extracted circular PRV DNA were prepared into transfection solution according to the following method:

washing BHK-21 cells growing up to 80% in a 6-well plate with opti-MEM medium for 2 times, adding all the mixed transfection solution to the washed cells, transfecting for 6 hours, replacing with DMEM medium containing 10% FBS, culturing for 48-72 hours, collecting cells and culture solution according to cell morphology, freeze-thawing for 3 times, inoculating all the collected virus solution into 1-well BHK-21 cells in a 6-well plate, culturing for 24-48 hours, and observing fluorescence generation and cytopathic effect under a fluorescence microscope. After 48 hours of culture, fluorescence produced by the cells was observed under a fluorescence microscope, and distinct cytopathic effects were observed at the corresponding fluorescence sites, the cells were rounded to form syncytia (see fig. 4), the virus fluid was harvested, frozen and thawed 3 times, and plaque-purified. Selecting stable fluorescent spots according to a virus plaque purification method, and continuously performing 5-7 rounds of purification to obtain a recombinant virus named as rPRV FB-delta gE/gI EGFP ⁺ :BAC ⁺ . Extracting recombinant virus genome DNA as a template, and carrying out PCR amplification by using PRV-gE-632-F and PRV-gE-632-R primers in table 1, wherein specific PCR amplification products cannot be amplified, and original strain DNA can amplify specific bands, so that the gE/gI gene is replaced by a corresponding sequence of a transfer plasmid, and the recombinant virus after plaque purification is free from wild virus pollutionSubsequent BAC construction or construction of a traceless deletion virus may be performed.

1.3 Construction of rPRV FB-delta gE/gI traceless Virus

Extracting rPRV FB-delta gE/gI EGFP ⁺ :BAC ⁺ The infectious genome of (4) was co-transfected with the transfer plasmid pUC19: H1: H2, the transfection method and the virus purification method were carried out as described in reference 1.2. Traceless recombinant viruses FB- Δ gE/gI (containing no fluorescence) with gE/I gene deletion and without EGFP and BAC sequences were screened by plaque purification techniques (FIG. 5) and virus stability and titer were determined.

Performing stable passage determination on FB-delta gE/gI recombinant virus, continuously performing passage for 20 generations, wherein cytopathic effect is consistent between each passage, performing titer determination on P1, P5, P10 and P20 generation viruses, and determining TCID thereof ₅₀ Are all at 10 ⁸ More than ml, the constructed deletion recombinant virus is proved to have good genetic stability.

Example 2 preliminary trial production of inactivated vaccine for gE/I Gene deletion of Pseudorabies virus FB Strain

2.1 Proliferation and inactivation of viruses

And (3) virus propagation: adopting a standing culture mode to carry out virus mass proliferation on the FB-delta gE/I deletion strain according to a conventional virus culture mode, carrying out freeze thawing for 3 times after proliferation, carrying out high-speed centrifugation at 15000rpm at 4 ℃ for 30min to remove protein fragments, collecting supernatant serving as a pre-inactivation antigen for later use, and measuring TCID according to a laboratory conventional method ₅₀ The virus stock solution was serially diluted 10-fold with serum-free DMED medium, and 10 cells were plated in a 96-well plate full of a monolayer of BHK21 cells ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 、10 ^-9 、10 ^-10 Respectively 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 cytopathy after PRV infection, and calculating TCID of virus according to Reed-Muench method ₅₀ . TCID of FB-delta gE/I deficient strain was determined ₅₀ Are respectively 10 ^8.6 /ml。

Antigen inactivation: adding appropriate amount of 10% slowly into the above antigen virus solutionThe final concentration of the formaldehyde is 0.1 percent, the formaldehyde is evenly shaken and then is completely transferred into a new sterile triangular flask to be inactivated for 36 hours at the temperature of 37 ℃, and the formaldehyde is continuously oscillated at 200rpm in the inactivation process to ensure that the formaldehyde is completely inactivated. After the inactivation, 5ml of inactivated virus solution was inoculated to 5 bottles of full monolayer BHK-21 cells (25 cm) ² ) And (4) continuously culturing for 7 days at 37 ℃ in each bottle of 1ml, observing whether cytopathic effect appears, and determining whether the virus is completely inactivated. The virus is completely inactivated by inspection.

2.2 Preparation of PRV FB-delta gE/I deletion strain inactivated vaccine

Preparing a proper amount of a two-phase adjuvant ISA 206VG (200 ml) and an aqueous phase antigen (200 ml) inactivated by the method of 2.1, respectively heating the two phases in a 31 ℃ water bath for 30 to 40min, placing a beaker filled with the adjuvant under a stirrer, stirring at the speed of 350rpm, taking out the heated aqueous phase antigen from the water bath, wiping the outer wall of the beaker, quickly adding an aqueous phase (about 3 s) into the adjuvant, stirring at the speed of 350rpm for 5min, stopping stirring and cooling, standing at 20 ℃ for 1 hour, and standing overnight at room temperature after the vaccine preparation is finished. After the preparation is finished, subpackaging, detecting the appearance and the quality of the subpackaged vaccine to meet the requirements, placing the vaccine in a glass bottle, storing at 4 ℃ and 20 ℃ respectively, and observing the stability to meet the requirements. .

Example 3 evaluation of immunogenicity of inactivated vaccine of PRV FB-. DELTA.gE/gI Strain

3.1 Test animals and grouping and handling

15 sheep weighing 15-20kg were divided into 3 groups of 5 sheep. The PRV FB-delta gE/I deletion inactivated vaccine prepared by using ISA 206VG adjuvant is used for immunizing sheep, the immunization mode is that the neck subcutaneous vaccine is 5ml, PRV FJ-2012 virulent strain is used after 4 weeks of immunization, the challenge dose is 1 ml/head, and the virus content is 10 ⁴ TCID ₅₀ . The groups and immunizations of the test animals are shown in Table 3.

TABLE 3 grouping and treatment of test animals

3.2 clinical observations

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.

3.3 results

The fourth day after the FJ-2012 strain is virulent and offensive, 5 sheep in the G2 group generate lying water samples, bite hind limbs, spit white foam, depression and fondness of lying, and 2 sheep die in the same day. No obvious clinical symptoms appeared in the groups G1 and G3.

The fifth day after challenge, 3 sheep in the G2 group all showed watery limbs, no stooping, and white foam in mouth, and all 3 sheep died in the same day. No obvious clinical symptoms appeared in the groups G1 and G3.

In the sixth to fourteen days after the toxin attack, no obvious clinical symptoms appear in the groups G1 and G3. Specific morbidity and mortality results are shown in table 4 and fig. 6.

Table 4. PRV FB-delta gE/I deletion inactivated vaccine prepared by ISA 206VG adjuvant protects rate of resisting PRV FJ-2012 virulent strain after immunization of sheep

ELISA detection is carried out on serum collected before and after sheep immunization, gB and gE antibodies of G1, G2 and G3 groups before the sheep immunization are all negative, and PRV infection is not shown. ELISA results of 7d, 14d, 21d and 28d after immunization show that gB antibodies of G1 group sheep are all positive, and gE antibodies are all negative, which shows that the vaccination effect is good, and PRV FB-delta gE/gI strain genes are completely deleted.

Example 4 comparison of the virulence of rabbits with different strains of PRV

4.1 Test animals and groups

20 normal grade healthy New Zealand rabbits weighing 1.5 kg were divided into 4 groups of 5 rabbits each.

4.2 Toxin counteracting toxic strain

FB strain, FB delta gE/gI strain and Bartha-K61 strain with the toxin counteracting dose of 10 ⁴ TCID ₅₀ The mode of toxic challenge was subcutaneous injection into the neck.

4.3 Clinical observations

After the challenge, the mental state and the morbidity of the animals are observed every day, whether the rabbits have respiratory symptoms and nervous symptoms or not and whether the inoculation part has bite and death or not are mainly observed, and the morbidity and mortality conditions are recorded.

4.4 Results

After continuously observing for 14 days after virus challenge, 3 rabbits of the FB strain survive, the FB delta gE/gI strain challenge group completely survives, but the Bartha-K61 strain group completely dies, so that the FB strain or the FB delta gE/gI strain has lower toxicity or virulence compared with the Bartha-K61 strain, which shows that the FB strain or the FB delta gE/gI strain has higher safety and can be used as a safe and effective attenuated vaccine candidate strain.

TABLE 5 mortality of different challenge strains in rabbits

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> construction of pseudorabies virus FB strain gE/gI gene deletion strain and application thereof as vaccine

<130> 16

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 1291

<212> DNA

<213> Artificial sequence

<400> 1

ttgggccctc tagatgcatg ctcgagcggc cgccagtgtg atggatatct gcagaattcg 60

cccttgtacc cgtacaccga gtcgtggcag ctgacgctga cgacggtccc ctcgcccttc 120

gtcggccccg cggacgtcta ccacacgcgc ccgctggagg acccgtgcgg ggtggtggcg 180

ctgatctccg acccgcaggt ggaccggctg ctgaacgagg cggtggccca ccggcggccc 240

acgtaccgcg cccacgtggc ctggtaccgc atcgcggacg ggtgcgcgca cctgctgtac 300

tttatcgagt acgccgactg cgaccccagg cagatctttg ggcgctgccg gcgccgcacc 360

acgccgatgt ggtggacccc gtccgcggac tacatgttcc ccacggagga cgagctgggg 420

ctgctcatgg tggccccggg gcggttcaac gagggccagt accggcgcct ggtgtccgtc 480

gacggcgtga acatcctcac cgacttcatg gtggcgctcc ccgaggggca agagtgcccg 540

ttcgcccgcg tggaccagca ccgcacgtac aagttcggcg cgtgctggag cgacgacagc 600

ttcaagcggg gcgtggacgt gatgcgattc ctgacgccgt tctaccagca gcccccgcac 660

cgggaggtgg tgaactactg gtaccgcaag aacggccgga cgctcccgcg ggcctacgcc 720

gccgccacgc cgtacgccat cgaccccgcg cggccctcgg cgggctcgcc gaggcccagg 780

cccaggcccc ggccccggcc ccggccgaag cccgagcccg ccccggcgac gcccgcgccc 840

cccggccgcc tgcccgagcc ggcgacgcgg gaccacgccg ccgggggccg ccccacgccg 900

cgacccccga ggcccgagac gccgcaccgc cccttcgccc cgccggccgt cgtgcccagc 960

gggtggccgc agcccgcgga gccgttcccg ccccggacca ccgccgcgcc gggcgtctcg 1020

cgccaccgct cggtgatcgt cggcacgggc accgcgatgg gcgcgctcct ggtgggcgtg 1080

tgcgtctaca tcttcttccg cctgaggggg gcgaaggggt atcgcctcct gggcggtccc 1140

gcggacgccg acgagctaaa agcgcagccc ggtccgtagc ctccgcagta ccggcgtcga 1200

tgatgaaagg gcgaattcca gcacactggc ggccgttact agtggatccg agctcggtac 1260

caagcttgat gcatagcttg agtattctat a 1291

<210> 2

<211> 1188

<212> DNA

<213> Artificial sequence

<400> 2

gccgacatgg acacgttcga ccccagcgcc cccgtcccga cgagcgtctc gaacccggcc 60

gccgacgtcc tgctggcccc caagggaccc cgctccccgc tgcgccccca ggacgactcg 120

gactgctact acagcgagag cgacaacgag acgcccagcg agttcctgcg ccgcgtggga 180

cgccggcagg cggcgcgtcg gagacgccgc cgctgcctga tgggcgtcgc gatcagcgcc 240

gccgcgctgg tcatctgctc gctgtccgcg ctactcgggg gcatcgtcgc caggcacgtg 300

tagcgagcga gcgaacggga gcgggggccc gctcccatcc gccgcgccca ggagaggggg 360

gagggcgcgg ggggttgagc gcgccacgtg gtgtgggcac ggactcggac ttgtcacaat 420

aaatgggccc cggcgcgtcc gggcgcacac agcagccttc ctctcctccg cgtctctgtt 480

ccgcccgtct ctcgccggac tcttcttctc caccgcctcc accgtcgcag ttgccgcgag 540

cgcgttcgca ccatgggggt gacggccatc accgtggtca cgctgatgga cggggccggg 600

cgcatccccg ccttcgtggg cgaggcgcac ccggacctgt ggaaggtgct caccgagtgg 660

tgctacgcgt cgatggtgca gcagcggcgc gccgccgacg agaactcgcc gcggcagcac 720

gtggtgctgc gctcctcgga gatctccccc ggctcgctgg ccctgctgcc gcgcgccgtg 780

cgccccgtcg tgcggacgcg gtccgacccc acggcgccgt tctacatcac caccgagacg 840

cacgagctga cgcggcgccc cccggcggac ggctcgaagc ccggggagcc cctccggatc 900

agcccgcccc cgcggctgga cacggagtgg tcgtccgtcc tgaacgggat ccagtacctg 960

aactcggggg cccggggcac ggcccccgtc cacctgtgga tcctgggtgc cgccgacctc 1020

tgcgaccagg tgctcctggc cgcctcccgc agcaccgccg ccggagcctc ccacgcccag 1080

acgggcgcgc gcctgacccg gcgccggccc gggctgacgg acgccgacgc cctggacgtg 1140

atcgtcgccg ggatccaggc gacccgcgcc atgttcgcgg gtccacaa 1188

<210> 3

<211> 1640

<212> DNA

<213> Artificial sequence

<400> 3

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600

ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660

gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 720

atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 780

gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 840

cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 900

taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 960

caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 1020

ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 1080

ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 1140

gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 1200

ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 1260

ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 1320

aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 1380

gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg 1440

tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa 1500

tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca 1560

atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 1620

ccaaactcat caatgtatct 1640

<210> 4

<211> 7507

<212> DNA

<213> Artificial sequence

<400> 4

gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 60

cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 120

gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg 180

aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg 240

caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 300

ccagtgaatt gtaatacgac tcactatagg gcgaattcga gctcggtacc cggggatcct 360

ctagagtcga cctgcaggca tgcaagcttg agtattctat agtgtcacct aaatagcttg 420

gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 480

aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 540

acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 600

cattaatgaa tcggccaacg cgaacccctt gcggccgccc gggccgtcga ccaattctca 660

tgtttgacag cttatcatcg aatttctgcc attcatccgc ttattatcac ttattcaggc 720

gtagcaacca ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg 780

ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca 840

aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata 900

tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa 960

actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt 1020

agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa 1080

ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg 1140

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

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> 16

gcacatcagc agcacatacg 20

Claims

1. An application of a gE/gI gene deletion strain of a pseudorabies virus FB strain in preparing a gE/gI gene deletion inactivated vaccine of the pseudorabies virus FB strain aiming at a new variation strain FJ-2012 is characterized in that: the gene deletion strain is named as a pseudorabies virus FB delta gE/gI strain, is preserved in China center for type culture Collection in 4 month and 8 days of 2020, and has the preservation number of CCTCC NO: v202026; the pseudorabies virus FB strain gE/gI gene deletion inactivated vaccine is a biphasic adjuvant inactivated vaccine, and can provide 100% protection for the challenge of a new variable strain FJ-2012.