CN113249341A

CN113249341A - Porcine pseudorabies virus double-gene deletion strain

Info

Publication number: CN113249341A
Application number: CN202110166079.5A
Authority: CN
Inventors: 孙伟; 刘杉杉; 高俊波; 朱锋钊; 段俊红
Original assignee: Tongren Polytechnic College
Current assignee: Tongren Polytechnic College
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2021-08-13

Abstract

The invention provides a recombinant porcine pseudorabies virus double-gene deletion strain, which is prepared by carrying out gE and gI gene deletion on a porcine pseudorabies virus TRZYT strain with the preservation number of CCTCC NO. 17403. The invention also provides a porcine pseudorabies virus vaccine which consists of an antigen and a protective agent, wherein the antigen is the porcine pseudorabies virus gene deletion strain prepared by the invention. The porcine pseudorabies virus gene deletion strain prepared by the invention is an attenuated strain, and has no virulence reversion phenomenon after continuous passage in a mouse body through horizontal propagation infection, has stable heredity, and meets the standard of the avirulence reversion of the porcine pseudorabies virus deletion vaccine strain. The vaccine prepared by the antigen can effectively prevent the porcine pseudorabies.

Description

Porcine pseudorabies virus double-gene deletion strain

Technical Field

The invention belongs to the technical field of preparation of attenuated vaccine strains, and particularly relates to a porcine pseudorabies virus double-gene deletion strain.

Background

Porcine Pseudorabies virus (PRV) belongs to the subfamily of herpesviridae, porcine herpesviridae, type I, in taxonomy, and mainly causes porcine Pseudorabies (PR). The pig is the only host of PRV, after PRV infects pregnant sow, abortion, stillbirth fetus and mummy fetus are mainly caused, sterility is mainly caused by infecting replacement sow, and nervous symptom and death are mainly caused by infecting piglet. The death rate of the porcine pseudorabies is even up to 100 percent, and the porcine pseudorabies seriously harms the pig industry in China.

At present, no effective medicine can treat the disease, and the prevention and control are mainly carried out by means of vaccination. In recent decades, pig farms have relied primarily on Bartha-K61 vaccine for control and have achieved significant success. However, since 2011, pig farms immunized with Bartha-K61 vaccine still have PRV infected pigs appeared in many provinces, and then are identified as variant PRV virulent strains. Namely, the existing Bartha-K61 vaccine can not effectively prevent and control the infection caused by PRV epidemic strains.

Therefore, the separated variant virulent strains need to be attenuated by natural attenuation or genetic engineering means to prepare vaccines for preventing and controlling PRV infection.

Disclosure of Invention

The invention aims to provide a recombinant porcine pseudorabies virus double-gene deletion strain and application thereof, namely deleting gE/gI genes of a porcine pseudorabies virus virulent strain to obtain an attenuated strain and preparing a vaccine.

The invention firstly provides a porcine pseudorabies virus gene deletion strain which is prepared by carrying out gE and gI gene deletion on a porcine pseudorabies virus TRZYT strain with the preservation number of CCTCC NO. 17403.

The invention also provides the application of the porcine pseudorabies virus gene deletion strain in preparing vaccines;

in another aspect, the invention provides a porcine pseudorabies virus vaccine, which consists of an antigen and a protective agent, wherein the antigen is the porcine pseudorabies virus gene deletion strain prepared by the invention.

Wherein the protective agent is an aqueous solution of sucrose and gelatin.

The porcine pseudorabies virus gene deletion strain prepared by the invention is an attenuated strain, and has no virulence reversion phenomenon after continuous passage in a mouse body through horizontal propagation infection, has stable heredity, and meets the standard of the avirulence reversion of the porcine pseudorabies virus deletion vaccine strain. The vaccine prepared by the antigen can effectively prevent the porcine pseudorabies.

Drawings

FIG. 1: the electrophoresis image of the PCR amplification product is shown in lane 1, lane 2, and lane 3, wherein lane 1 is marker, lane 2 is a negative control of ultrapure water, and lane 3 is a virus collection solution.

FIG. 2: the isolated culture picture of the virus TRZYT strain, wherein the left picture is a normal Vero cell, and the right picture is a cell which is inoculated with pathological materials and has cytopathic effect;

Detailed Description

On the basis of screening to obtain a porcine pseudorabies virus virulent strain TRZYT strain with genetic variation, the gE gene and the gI gene of the TRZYT strain are deleted by adopting the existing method to prepare an attenuated strain, and the prepared attenuated strain can provide effective immune protection for the screened variant TRZYT strain.

The gE gene of PRV is essential for the invasion of PRV from retina, olfactory epithelium and trigeminal ganglion into central nervous tissue. The gE gene plays a decisive role in the virulence expression, nerve invasion and nerve transmission of PRV (porcine pseudorabies virus), while the gE gene deletion strain of PRV can only invade the first level of the nervous system, so that the toxicity of PRV is weakened due to gE gene deletion.

The gI gene is located in front of the gE gene, and deletion thereof affects the neurotropic nature of PRV virus. PRV deletion of gE and gI genes does not affect retrograde viral propagation, but limits anterograde viral propagation in neurons, thereby reducing toxicity of PRV viruses.

The present invention is further described with reference to the following specific embodiments, and a person skilled in the art may select the method steps commonly used in the art based on the technical solution of the present invention, and not limited to the specific descriptions of the embodiments in the present specification.

Example 1: screening and identification of porcine pseudorabies virus

1. Screening for viral strains

In 11-month Guizhou-province pig farm in 2018, a pig herd injected with a porcine pseudorabies live vaccine (Bartha-K61 strain vaccine) has a porcine pseudorabies disease, and porcine pseudorabies viruses are screened from the diseased pig herd. The specific screening steps are as follows:

taking a liver and lymph node sample of a diseased pig, homogenizing the sample by using 0.1M PBS (phosphate buffer solution) with the pH value of 7.2, repeatedly freezing and thawing, centrifuging for 10min at 5000r/min, taking supernatant, adding 10 mu L of streptomycin, standing for 1h at 37 ℃, and filtering and sterilizing by using a 0.22 mu M filter membrane. Adding Vero cells (figure 1) which just grow into a monolayer into 10 mu L of filtrate, collecting toxicity after pathological changes appear, and repeatedly freezing and thawing at-80 ℃ for detection.

2. Detection of viruses

Amplifying PRV gE gene fragments by using a primer pair with the following sequences, amplifying the collected virus liquid by using Oligo 7.0 design primers, and carrying out electrophoresis on PCR amplification products, wherein the virus liquid is amplified to form a target band (figure 2); the sequence information of the amplification primers is as follows:

F1:5′-ATGCGGCCCTTTCTGCTGCGCGCC-3′；

F2:5′-TTAAGCGGGGCGGGACATCAACAGGC-3′。

the screened porcine pseudorabies virus is named as TRZYT strain, and the virus content reaches 10 after the cultivation and domestication of Vero cells^7.0TCID₅₀More than 0.1 ml.

3. Detection of virulence of a virus

Mixing TRZYT strain virus liquid (virus content is 10)^7.5TCID₅₀0.1ml) were dispensed and the titer was determined at-80 ℃ for 36 months. The virus content was determined to be 10^7.1TCID₅₀0.1 ml. The result shows that the strain has better stability.

10 BALB/c mice 6 weeks old were randomly divided into two groups of 5 mice each, and 200TCID was tried₅₀The diluted virus was injected subcutaneously into the neck, at an inoculum size of 0.5 mL/tube. The other group was inoculated with an equal amount of PBS solution as a negative control. The clinical symptoms and mortality of the mice were observed and recorded daily. Results mice inoculated with this virus fluid exhibited typical pseudorabies symptoms: the sick mice bite the inoculation part, severe skin bleeding and fur shedding occur, and the mice inoculated with the virus group die after 120 hours of inoculation. The control mice had no abnormal changes. The virus strain is indicated to be a virulent strain.

4. Animal regression test

After 4-8 days old PRV antibody negative piglets are inoculated and injected for 48-72 hours, obvious clinical symptoms are presented, which mainly show that the body temperature is raised, the gait is unstable, the milk intake is reduced, the movement is not coordinated, the four limbs are in a water-scratching shape, and partial pigs vomit or diarrhea and other symptoms. The brain membrane congestion, the cerebrospinal fluid amount is too much, and the necrosis of organs such as the liver, the spleen and the like can be found through dissection; and the PRV strain can be re-isolated from brain tissue.

5. Antibody neutralization assay

TRZYT strain and Porcine pseudorabies virus QD strain (herpes virus Type I, Portone herpesvirus Type I, preservation number is CGMCC No.10266, which has been preserved in the general microbiological center of China institute of microbiology, China institute of sciences, China, institute of microbiology, No.1, North Chen West Lu, No. 3, of the sunward, Beijing city for 3 months and 6 days in 2015) are respectively subjected to subculture and purification to prepare inactivated vaccines, mice are respectively immunized with the inactivated vaccines in an amount of 2.0 ml/mouse, the mice are immunized once every 2 weeks for 3 times, blood is collected 2 weeks after the third immunization, serum is separated, and neutralization experiments are performed after inactivation at 50 ℃.

Diluting TRZYT strain virus liquid by 100 times, mixing with blood serum prepared from TRZYT strain and porcine pseudorabies virus QD strain respectively in equal volume, neutralizing at room temperature for 1 hour, inoculating to mouse, and observing for 96-168 hours after inoculation.

The result shows that no disease occurs in the mice in the TRZYT strain serum neutralization group of the TRZYT strain, and 30-40% of the mice in the porcine pseudorabies virus QD strain serum neutralization group have the porcine pseudorabies disease. The neutralizing capacity of the positive serum prepared by using the strain screened by the invention as an antigen to the positive serum is obviously better than that of the positive serum prepared by using other strains; the TRZYT virus strain obtained by the invention is a new porcine pseudorabies virus variant strain, so that the immune effect of the existing vaccine is reduced.

6. Analysis of antigen genes

The gB, gC, gD and gI genes of the TRZYT strain and the QD strain of the porcine pseudorabies virus are amplified and sequenced respectively, and the four genes of the TRZYT strain and the QD strain are analyzed, so that the result shows that the immune genes of the RZYT strain and the QD strain of the porcine pseudorabies virus have amino difference, and the TRZYT strain is shown to be mutated compared with the prior QD strain of an epidemic strain (Table 1); this is presumably the reason why the neutralizing effect of the antibody serum prepared using the QD strain as an antigen on the TRZYT strain is not good.

Table 1: amino acid sequence difference table of virulence genes of four porcine pseudorabies viruses of TRZYT strain and QD strain

The result of the challenge protection test shows that the TRZYT strain inactivated vaccine can provide complete immune protection for TRZYT strain virus, and partial protection for QD strain virus. The QD strain inactivated vaccine can completely protect the QD strain virus and only partially protect the TRZYT strain virus. The Bartha-K61 strain vaccine has poor protection effect on TRZYT strain and QD strain. This indicates that the cross protection between the TRZYT strain and the QD strain is poor, and also indicates that the TRZYT strain isolated by the invention is a new variant strain of porcine pseudorabies virus.

Example 2: construction of recombinant porcine pseudorabies virus gE/gI double-gene deletion strain

Extracting DNA of the separated strain from the separated porcine pseudorabies virus TRZYT strain, and carrying out the deletion of virulence genes gE and gI on the separated strain by adopting a genetic engineering method.

Reference to PRV whole genome sequence (BK001744) was made to synthesize a series of primers for amplifying the left arm fragment (L) and right arm fragment (R) flanking (containing part of the gI and gE genes) the US7(gI) gene and US8(gE) gene, respectively, which can be used for homologous recombination. Wherein L comprises a part of the US6 gene and a part of the gI gene, and R comprises a part of the gE gene, the whole US9 gene and a part of the US2 gene.

In addition, primers are designed to amplify EGFP and EGFP eukaryotic expression cassettes and used for identifying gene deletion. The primer sequences and the sizes of the expected PCR products are shown in Table 2.

Table 2: primer sequence information Table

2. Construction of gE and gI Gene transfer vectors

Taking TRZYT strain genome as a template, and utilizing primers gEILF/GEILR and gEIRF/GEIRR to respectively amplify sequences (containing partial gE and gI genes) positioned at two sides of gE and gI to be used as a left recombination arm gEIL and a right recombination arm gEIR, wherein one end of the gEIL and one end of the gEIR are respectively provided with a loxP site. Cloning the fragment into pMD19-T, carrying out PCR and enzyme digestion identification on the fragment, identifying the fragment correctly, sequencing the fragment, carrying out double enzyme digestion on a positive clone T-gEIL with correct sequencing by adopting Hind III and Pst I, cloning the fragment onto a pBluescript SK vector subjected to the same enzyme digestion treatment, identifying the fragment correctly, carrying out double enzyme digestion on the recombinant plasmid and the recombinant plasmid vector connected with the right homologous arm by adopting Spe I and Xba I respectively, recovering a linearized recombinant plasmid containing the left homologous arm and the right homologous arm respectively, and connecting the two. And performing PCR and enzyme digestion for identification. The sequencing is carried out after the identification is correct, and the sequencing result is correct and is named as pSKgEILR.

The method comprises the steps of taking pCDNA 3.1-EGFP plasmid as a template, adopting a primer EorfF/EorfR to amplify an EGFP reading frame, connecting the EGFP reading frame with a eukaryotic expression vector pVAX1, adopting a primer cassetteF/cassetteF to amplify an EGFP-containing eukaryotic expression cassette after correct identification, connecting the EGFP-containing eukaryotic expression cassette with a pMD19-T vector, and naming the EGFP-containing eukaryotic expression cassette as pMDEV after correct identification.

Carrying out double enzyme digestion on pMDEV by adopting Pst I and Spe I, recovering a eukaryotic expression cassette, connecting pSKgEILR subjected to the same double enzyme digestion, and obtaining a gE and gI gene transfer vector after correct identification, wherein the gE and gI gene transfer vector is named as pSKgE-EGFP.

3. Construction and purification rescue of gE and gI gene deletion strain virus

3.1 transfection

Recombinant virusesRescue was performed on six-well cell culture plates and transfection was performed when Vero cells grew to 90%. Taking 3 mu g of TRZYT strain genome, mixing with 1 mu g of transfer plasmid pSKgE-EGFP, and co-transfecting according to a conventional method, wherein the detailed method is Lipofectamine^TM2000, specification. A control group containing only the transfer plasmid was also prepared.

3.2, identification

After the TRZYT strain genome and pSKgE-EGFP are co-transfected for 48 hours, the lesion formation condition and the fluorescent protein expression condition of the transfected cells are observed. After the transfected cells are lysed, the cells are transmitted for two blind generations, and cytopathic effect and green fluorescence are still remained, so that the success of the rescue of the recombinant viruses is preliminarily judged. The recombinant virus is identified by plaque purification and PCR and is named PRV/gE^-/gI^-/EGFP。

3.3 deletion of reporter Gene of deleted Strain

And (3) knocking out the reporter gene EGFP by utilizing a Cre-loxP system mediated site-specific recombination technology. 10 ug of Cre enzyme treated DNA was transfected into Vero cells by calcium phosphate method and placed at 37 ℃ in CO₂Culturing in incubator for 2-3 days, and collecting virus liquid when 80% of cells generate lesions. And (3) freeze thawing the virus liquid for three times repeatedly, taking the supernatant, inoculating the supernatant into Vero cells grown into a single layer in a 24-pore plate according to 2 uL/pore, paving low-melting-point agarose when cytopathic effect begins to appear at first, and picking out the plaques without fluorescence under a fluorescence microscope. The viruses are purified repeatedly until all virus plaques do not have green light burst, and the constructed TRZYT strain double-gene deletion strain is named as PRV/gE^-/gI^-。

3.4 double Gene deletion value PRV/gE^-/gI^-Identification of

Extraction of PRV/gE^-/gI^-Genomic DNA, PCR amplification with primers gEIF/gEIR and gBF/gBR to determine whether the rescued viral gene internal fragment is deleted, parental strain, PRV/gE^-/gI^-EGFP genomic DNA was used as a control.

Successfully obtaining a gE and gI gene transfer vector by taking a porcine pseudorabies virus TRZYT strain as a template; then carrying out homologous recombination on the EGFP gene and PRV genome to successfully rescue and purify gE and gI gene deletion strain viruses containing EGFP marker genes; using Cre recombinaseAfter the EGFP marker gene is removed, gE and gI gene deletion strain viruses (PRV/gE) without the EGFP marker gene are successfully rescued and purified^-/gI^-)。

4. Genetic stability testing of recombinant viruses

Primary generation PRV/gE obtained by screening^-/gI^-The recombinant virus is continuously passaged on Vero cells, total DNA of infected cells is extracted every 5 generations, and PCR detection of deletion part genes is carried out.

The PRV/gE obtained by screening^-/gI^-The primary recombinant virus is continuously passaged on Vero cells, total DNA of infected cells is extracted every 5 generations, and the PCR detection result of the deletion part of genes shows that the sizes of the genes are the sizes after deletion fragments, so that the stability of the recombinant virus is good.

5、PRV/gE^-/gI^-Safety test of virus seeds

The antigen was diluted 10-fold with PBS, and four mice 100 g, 0.2mL each, were intramuscularly inoculated, and observed for 14 days, which had to be more than two in response or death.

The result of the virus seed safety test shows that the vaccine is safe to mice, has no pseudorabies specific symptoms and does not influence growth and development. Therefore, the porcine pseudorabies virus gene deletion vaccine (PRV/gE-/gI-) obtained by the invention is safe and can be used for preparing vaccines.

6、PRV/gE^-/gI^-(ii) detection of immunopotency

The 6-week-old BALB/C mice were randomly divided into 3 groups of 5 mice each and weighed, and injected with vaccine strain Bartha-K61 and deletion strain PRV/gE, respectively^-/gI^-And DMEM. Each control group was injected with 100. mu.L of DMEM, and the other two groups were injected with 10. mu.L of hind limb muscle⁴TCID₅₀A viral dose of the vaccine. After immunization, the mice were observed daily for clinical symptoms of listlessness, anorexia, pruritus, tremor, etc., and were weighed again at 21d, and blood was collected at 14d and 21d tail veins, and serum was isolated and tested for serum antibody levels using an ELISA plate coated with TRZYT strain whole virus. After immunization 21d, the TRZYT strain is used for counteracting the toxin, and the dose is 10⁴TCID₅₀The dose of the virus is inoculated to each test group and control group, the injection is carried out by hind limb muscle, and the virus is continuously inoculatedAnd (5) observing 14 d.

Post-immunization 21d, Bartha-K61, PRV/gE^-/gI^-The average weight gain of mice in the group and the DMEM control group is respectively 4.4g, 4.1g and 4.5g, the weight gain difference is small, and the result shows that the deletion strain has no inhibition effect on the weight gain of the mice. No antibody was detected at 14d after immunization, and PRV/gE at 21d^-/gI^-The antibody levels in the groups were significantly elevated and were not significantly changed in the other groups. Indicating PRV/gE^-/gI^-The deletion strain can induce mice to generate obvious immune response. After 21d immunization, TRZYT strain is used for counteracting toxic pathogen, PRV/gE^-/gI^-The group protection rate is obviously higher than that of Bartha-K61 group, and the protection rates are 100% and 50% respectively.

Example 3 preparation and application of porcine pseudorabies virus gene deletion vaccine

Vero cells were cultured according to the conventional method with PRV/gE^-/gI^-Infecting cells with the virus strain, harvesting the virus when cytopathic effect (CPE) reaches about 90%, repeatedly freezing and thawing at-80 deg.C for three times, and determining the TCID of the virus₅₀. And (3) according to the poison price of the harvested virus liquid, carrying out proper dilution, wherein the volume ratio of the diluted virus liquid to the protective agent is 1:1.5, and fully mixing, wherein the final concentration of the sucrose is 20%, and the final concentration of the gelatin is 4.8%. 2.5mL of the solution was taken in each bottle and lyophilized in a lyophilizer. The content of the porcine pseudorabies virus in the vaccine is 10^6.5TCID₅₀/0.1ml。

1. Inspection of finished product

1) Traits

Observing the appearance color and character of the vaccine, the separation condition of the vaccine from the bottle wall and the dissolution condition of the vaccine after adding the diluent. The yellowish spongy loose lumps are easy to separate from the bottle wall when the sample is shaken up and down. The diluent is added to dissolve rapidly.

2) Sterility testing

The test was carried out according to the method of appendix of the 2010 version of Chinese pharmacopoeia. The vaccine was randomly sampled from 10 bottles, the original amount was recovered with 10% DMEM, and each bottle was examined according to the method of addendum of the 2010 version of the chinese pharmacopoeia. No bacteria and mould grow.

3) Mycoplasma assay

The test was carried out according to the method of appendix of the 2010 version of Chinese pharmacopoeia. The vaccines were randomly sampled in 5 bottles, restored to original amounts with 10% DMEM, and mixed, and examined according to the method of addendum of the 2010 version "chinese animal pharmacopoeia". None of the vaccines grew as mycoplasma.

4) Exogenous virus assay

The test was carried out according to the method of appendix of the 2010 version of Chinese pharmacopoeia. And extracting a cell virus genome, and performing PCR identification on the exogenous virus, wherein the results are negative, which indicates that the vaccine exogenous virus is qualified in inspection.

5) Safety inspection

After 10 BALB/C mice of 6 weeks old are injected intramuscularly with 0.2ml (containing 10 feathers) of vaccine, the mice are observed for 21 days and are all healthy and alive without any local or systemic adverse reaction.

The vaccine was sampled from 3 bottles, and the samples were recovered with 10% DMEM, diluted appropriately, and each was injected intramuscularly with 0.2ml of the vaccine for 21 days. The results show that the inoculated mice have no adverse reaction, and 10/10 is healthy and alive.

6) Efficacy test

According to the feather marked by the bottle label, the vaccine is diluted to l feather/0.2 ml by 10 percent DMEM, and then 10 times of serial dilution is carried out, and 10 times of the serial dilution is taken^-2、10^-3、10^-4、10^-5And respectively inoculating Vero cells with good growth state in four dilutions, inoculating 8 wells with 0.2m1 in each dilution, and inoculating 10% DMEM in another 8 wells as a control. Incubation of CO at 37 deg.C₂After 3 days of incubation in the incubator, the pathological changes were observed and TCID was calculated according to the Reed-Muench method₅₀The virus content should be more than or equal to 10^4.0TCID₅₀/0.2ml。

According to the feather marked by the bottle label, diluting the vaccine to l feather/0.2 ml by 10% DMEM, then performing 10-time serial dilution, and calculating TCID (TCID) according to a Reed-Muench method after inoculating cells₅₀The results show that each plume is 10^4.5TCID₅₀. None of the control mice had the specific clinical symptoms of pseudorabies.

10 BALB/C mice of 6 weeks old were injected with 1 feather of vaccine per muscle. After 21 days, along with 10 control mice, hind limb muscle injection 10^4.0TCID₅₀14 days of observation, 10 deaths from the control groupDeath or appearance of pseudorabies-specific symptoms.

7) Residual moisture determination

The method is carried out according to an annex method of Chinese animal pharmacopoeia 2010 edition. Vaccine sampling 4 vials were tested by vacuum drying. And the result shows that the residual water content of the sample is 2.0-2.7% and is less than or equal to 4%. The vaccine residual water content is qualified in the determination test.

8) Measurement of vacuum degree

The method is carried out according to an annex method of Chinese animal pharmacopoeia 2010 edition. The vaccines were separately tested with a vacuum leak detector. Results the test samples all glow purple. The vacuum degree measurement and the inspection of the vaccine are all qualified.

9) Vaccine use effect test

Selecting 15 piglets of 30 days old before test, randomly dividing into 3 groups, weighing 5 piglets in each group, and injecting vaccine strain Bartha-K61 and deletion strain PRV/gE respectively^-/gI^-Vaccine and DMEM. Each control group was injected with 2mL DMEM, and the other two groups were injected with 10 mL of hind limb muscle^5.0TCID₅₀A viral dose of the vaccine. Using porcine pseudorabies virus TRZYT strain with the preservation number of CCTCC NO.17403 to perform virus challenge at 4 weeks after immunization, wherein the dosage is 10^7.0TCID₅₀And observing clinical symptoms and calculating the protection rate condition.

The result of toxic attack shows that PRV/gE^-/gI^-The group protection rate is obviously higher than that of Bartha-K61 group (the protection rate of the Bartha-K61 group to the TRZYT strain is not higher than 60 percent), which shows that the PRV/gE of the invention^-/gI^-The clinical immune effect of the vaccine is obviously better than that of the Bartha-K61 vaccine widely used at present. Moreover, the detection effect shows that the immunization effect of the PRV/TK-/gE-vaccine on the TRZYT strain used by the invention is obviously better than that of other vaccines (p is less than 0.05); prove that the porcine pseudorabies virus TRZYT strain serving as the initial strain has genetic specificity.

Sequence listing

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900 905 910

Ala Pro

<210> 2

<211> 487

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Ser Leu Ala Arg Ala Met Leu Ala Leu Leu Ala Leu Tyr Thr

1 5 10 15

Ala Ala Ile Ala Ala Ala Pro Ser Ser Thr Thr Ala Leu Gly Thr Thr

20 25 30

Pro Asn Gly Gly Gly Gly Gly Asn Ser Ser Ala Gly Glu Leu Ser Pro

35 40 45

Ser Pro Pro Ser Thr Pro Glu Pro Val Ser Gly Thr Thr Gly Ala Ala

50 55 60

Ala Ser Thr Pro Ala Ala Val Ser Thr Pro Arg Val Pro Pro Pro Ser

65 70 75 80

Val Ser Arg Arg Lys Pro Gln Arg Asn Gly Asn Arg Thr Arg Val His

85 90 95

Gly Asp Lys Ala Thr Ser His Gly Arg Lys Arg Ile Val Cys Arg Glu

100 105 110

Arg Leu Phe Ser Ala Arg Val Gly Asp Ala Val Ser Phe Gly Cys Ala

115 120 125

Val Val Pro Arg Ala Gly Glu Thr Phe Glu Val Arg Phe Cys Arg Arg

130 135 140

Gly Arg Phe Arg Ser Pro Asp Ala Asp Pro Glu Tyr Phe Asp Glu Pro

145 150 155 160

Pro Arg Pro Glu Leu Pro Arg Glu Arg Leu Leu Phe Ser Ser Ala Asn

165 170 175

Ala Ser Leu Ala His Ala Asp Ala Leu Ala Ser Ala Val Val Val Glu

180 185 190

Gly Glu Arg Ala Thr Val Ala Asn Val Ser Gly Glu Val Ser Val Arg

195 200 205

Val Ala Ala Ala Asp Ala Glu Thr Glu Gly Val Tyr Thr Trp Arg Val

210 215 220

Leu Ser Ala Asn Gly Thr Glu Val Arg Ser Ala Asn Val Ser Leu Val

225 230 235 240

Leu Tyr His Gln Pro Glu Phe Gly Leu Ser Ala Pro Pro Val Leu Phe

245 250 255

Gly Glu Pro Phe Arg Ala Val Cys Val Val Arg Asp Tyr Tyr Pro Arg

260 265 270

Arg Ser Val Arg Leu Arg Trp Phe Ala Asp Glu His Pro Val Asp Ala

275 280 285

Ala Phe Val Thr Asn Ser Thr Val Ala Asp Glu Leu Gly Arg Arg Thr

290 295 300

Arg Val Ser Val Val Asn Val Thr Arg Ala Asp Val Pro Gly Leu Ala

305 310 315 320

Ala Ala Asp Asp Ala Asp Ala Leu Ala Pro Ser Leu Arg Cys Glu Ala

325 330 335

Val Trp Tyr Arg Asp Ser Val Ala Ser Gln Arg Phe Ser Glu Ala Leu

340 345 350

Arg Pro His Val Tyr His Pro Ala Ala Val Ser Val Arg Phe Val Glu

355 360 365

Gly Phe Ala Val Cys Asp Gly Leu Cys Val Pro Pro Glu Ala Arg Leu

370 375 380

Ala Trp Ser Asp His Ala Ala Asp Thr Val Tyr His Leu Gly Ala Cys

385 390 395 400

Ala Glu His Pro Gly Leu Leu Asn Val Arg Ser Ala Arg Pro Leu Ser

405 410 415

Asp Leu Asp Gly Pro Val Asp Tyr Thr Cys Arg Leu Glu Gly Met Pro

420 425 430

Ser Gln Leu Pro Ile Phe Glu Asp Thr Gln Arg Tyr Asp Ala Ser Pro

435 440 445

Thr Ser Val Ser Trp Pro Val Val Thr Ser Met Ile Thr Val Ile Ala

450 455 460

Gly Ile Ala Ile Leu Ala Ile Val Leu Val Ile Met Ala Thr Cys Val

465 470 475 480

Tyr Tyr Arg Arg Ser Ala Leu

485

<210> 3

<211> 402

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr Thr Leu

1 5 10 15

Gly Ala Asp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala

20 25 30

Tyr Pro Tyr Thr Glu Ser Trp Gln Leu Thr Leu Thr Thr Val Pro Ser

35 40 45

Pro Phe Val Gly Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu Asp

50 55 60

Pro Cys Gly Val Val Ala Leu Ile Ser Asp Pro Gln Val Asp Arg Leu

65 70 75 80

Leu Asn Glu Ala Val Ala His Arg Arg Pro Thr Tyr Arg Ala His Val

85 90 95

Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu Leu Tyr Phe Ile

100 105 110

Glu Tyr Ala Asp Cys Asp Pro Arg Gln Ile Phe Gly Arg Cys Arg Arg

115 120 125

Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro

130 135 140

Thr Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn

145 150 155 160

Glu Gly Gln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu

165 170 175

Thr Asp Phe Met Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala

180 185 190

Arg Val Asp Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp

195 200 205

Asp Ser Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro Phe

210 215 220

Tyr Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg Lys

225 230 235 240

Asn Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala Thr Pro Tyr Ala

245 250 255

Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg Pro Arg

260 265 270

Pro Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala Pro Ala Thr Pro

275 280 285

Ala Pro Pro Gly Arg Leu Pro Glu Pro Ala Thr Arg Asp His Ala Ala

290 295 300

Gly Gly Arg Pro Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg

305 310 315 320

Pro Phe Ala Pro Pro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala

325 330 335

Glu Pro Phe Pro Pro Arg Thr Thr Ala Ala Pro Gly Val Ser Arg His

340 345 350

Arg Ser Val Ile Val Gly Thr Gly Thr Ala Met Gly Ala Leu Leu Val

355 360 365

Gly Val Cys Val Tyr Ile Phe Phe Arg Leu Arg Gly Ala Lys Gly Tyr

370 375 380

Arg Leu Leu Gly Gly Pro Ala Asp Ala Asp Glu Leu Lys Ala Gln Pro

385 390 395 400

Gly Pro

<210> 4

<211> 366

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Met Met Val Ala Arg Asp Val Thr Arg Leu Pro Ala Gly Leu Leu

1 5 10 15

Leu Ala Ala Leu Thr Leu Ala Ala Leu Thr Pro Arg Val Gly Gly Val

20 25 30

Leu Phe Arg Gly Ala Gly Val Ser Val His Val Ala Gly Ser Ala Val

35 40 45

Leu Val Pro Gly Asp Ala Pro Asn Leu Thr Ile Asp Gly Thr Leu Leu

50 55 60

Phe Leu Glu Gly Pro Ser Pro Ser Asn Tyr Ser Gly Arg Val Glu Leu

65 70 75 80

Leu Arg Leu Asp Pro Lys Arg Ala Cys Tyr Thr Arg Glu Tyr Ala Ala

85 90 95

Glu Tyr Asp Leu Cys Pro Arg Val His His Glu Ala Phe Arg Gly Cys

100 105 110

Leu Arg Lys Arg Glu Pro Leu Ala Arg Arg Ala Ser Ala Ala Val Glu

115 120 125

Ala Arg Arg Leu Leu Phe Val Ser Arg Pro Ala Ser Gly Asp Ala Gly

130 135 140

Ser Tyr Val Leu Arg Val Arg Val Asn Gly Thr Thr Asp Leu Phe Val

145 150 155 160

Leu Thr Ala Leu Val Pro Pro Arg Gly Arg Pro Val Pro Thr Ser Pro

165 170 175

Pro Ala Asp Glu Cys Arg Pro Val Val Gly Ser Trp His Asp Ser Leu

180 185 190

Arg Val Val Asp Pro Ala Glu Asp Ala Val Phe Thr Thr Gln Pro Pro

195 200 205

Pro Glu Pro Glu Pro Pro Thr Thr Pro Ala Pro Pro Arg Gly Thr Gly

210 215 220

Ala Thr Pro Glu Pro Arg Ser Asp Glu Glu Glu Glu Gly Asp Ala Glu

225 230 235 240

Thr Thr Thr Pro Thr Leu Thr Pro Ala Pro Gly Thr Leu Asp Ala Asn

245 250 255

Gly Thr Met Val Leu Asn Ala Ser Val Val Ser Arg Val Leu Leu Ala

260 265 270

Ala Ala Asn Ala Thr Ala Gly Ala Arg Gly Pro Gly Lys Ile Ala Met

275 280 285

Val Leu Gly Pro Thr Ile Val Val Leu Leu Ile Phe Leu Gly Gly Ile

290 295 300

Ala Cys Val Ala Arg Arg Cys Ala Arg Asn Arg Ile Tyr Arg Pro Arg

305 310 315 320

Pro Gly Arg Gly Ser Ala Val His Ala Ala Pro Pro Arg Arg Pro Pro

325 330 335

Pro Asn Pro Val Ala Gly Ala Pro Val Pro Gln Pro Lys Met Thr Leu

340 345 350

Ala Glu Leu Arg Gln Lys Leu Ala Thr Ile Ala Glu Glu Gln

355 360 365

Claims

1. The porcine pseudorabies virus gene deletion strain is characterized by being prepared by carrying out gE and gI gene deletion on porcine pseudorabies virus with the preservation number of CCTCC NO. 17403.

2. The use of the porcine pseudorabies virus gene deletion strain of claim 1 in the preparation of a vaccine.

3. A porcine pseudorabies virus vaccine, characterized in that the antigen of the vaccine is the porcine pseudorabies virus gene deletion strain of claim 1.

4. The porcine pseudorabies virus vaccine according to claim 3, wherein said vaccine further comprises a protective agent.

5. The porcine pseudorabies virus vaccine according to claim 4, wherein the protective agent is an aqueous solution of sucrose and gelatin.