CN113151190B

CN113151190B - Porcine pseudorabies virus virulent strain

Info

Publication number: CN113151190B
Application number: CN202110106250.3A
Authority: CN
Inventors: 刘杉杉; 孙伟; 曾泽; 张稳; 周佳
Original assignee: Tongren Polytechnic College
Current assignee: Tongren Polytechnic College
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2023-10-31
Anticipated expiration: 2041-01-26
Also published as: CN113151190A

Abstract

The application provides a porcine pseudorabies virus virulent strain, which is a porcine pseudorabies virus TRZYT strain and is preserved in China general microbiological culture Collection center (CCTCC) No.17403, which is located in the North western road of the Korean region of Beijing, 3 months and 15 days in 2019. The application of the pseudorabies virus in preparing the porcine pseudorabies virus attenuated strain is provided; the application provides application of a pseudorabies virus TRZYT strain in preparation of an inactivated vaccine. Compared with the reported porcine pseudorabies virus, the porcine pseudorabies virus TRZYT strain screened by the application has certain variation, the prepared vaccine can induce the generation of neutralizing antibodies, and compared with the inactivated vaccine prepared by other porcine pseudorabies viruses, the vaccine prepared by the TRZYT strain has the best immune effect on the porcine pseudorabies virus.

Description

Porcine pseudorabies virus virulent strain

Technical Field

The application belongs to the technical field of vaccine virus strain screening, and particularly relates to a porcine pseudorabies virus virulent strain.

Technical Field

Porcine Pseudorabies virus (Pseudorabies virus, PRV) belongs to the taxonomic subfamily alphaherpesviridae porcine herpesvirus type I, which mainly causes Pseudorabies (PR) in pigs. Pigs are the only host of PRV, after PRV infection of pregnant sows, abortion, stillbirth, mummy and the like are mainly caused, infection of replacement gilts is mainly caused to cause infertility, and infection of piglets is mainly caused to cause neurological symptoms, death and the like. The death rate of the pseudorabies of pigs is even up to 100 percent, and the pig industry in China is seriously endangered.

At present, no effective medicine can treat the disease, and the disease is prevented and controlled mainly by vaccination. In recent decades, pig farms have been controlled mainly by means of the Bartha-K61 vaccine, with remarkable results. However, since 2011, pigs still have been infected with PRV in pig farms immunized with Bartha-K61 vaccine, and were identified as variant PRV virulent strains. That is, the existing Bartha-K61 vaccine can not effectively prevent and control infection caused by PRV epidemic strains. Therefore, it is necessary to prepare a vaccine from the isolated variant virulent strain by natural attenuation or genetic engineering means to prevent and control PRV infection.

However, with the mutation of the porcine pseudorabies epidemic disease in China, the existing vaccine cannot completely protect the attack of the mutated porcine pseudorabies virus. Therefore, the isolation of variant strains of porcine pseudorabies virus and deletion of virulence genes thereof to obtain vaccine strains has been the focus of research in the field of porcine pseudorabies virus.

Disclosure of Invention

The application aims to provide a porcine pseudorabies virus virulent strain, so as to make up for the defects of the prior art.

The porcine pseudorabies virus strain provided by the application is a porcine pseudorabies virus TRZYT strain, and is preserved in China general microbiological culture Collection center (CGMCC NO. 17403) of the North-Chen west road in the Korean region of Beijing in 3 months and 15 days.

The application of the pseudorabies virus in preparing the porcine pseudorabies virus attenuated strain is provided;

the application provides application of a pseudorabies virus TRZYT strain in preparation of an inactivated vaccine.

Compared with the reported porcine pseudorabies virus, the porcine pseudorabies virus TRZYT strain screened by the application has certain variation, the prepared vaccine can induce the generation of neutralizing antibodies, and compared with the inactivated vaccine prepared by other porcine pseudorabies viruses, the vaccine prepared by the TRZYT strain has the best immune effect on the porcine pseudorabies virus.

Drawings

Fig. 1: the virus TRZYT strain is isolated and cultured, wherein the left graph is normal Vero cells, and the right graph is cells inoculated with a disease agent and showing cytopathy;

fig. 2: detection electrophoretogram of PCR amplified product, wherein lane 1 is marker, lane 2 is negative control of ultrapure water, and lane 3 is virus collection liquid.

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The following examples are intended to further illustrate the specific manner in which the application may be practiced and should not be construed as limiting the application. Modifications of the application will fall within the scope of the appended claims without departing from the spirit and principles of the application.

The present application will be described in detail with reference to the following examples and the accompanying drawings.

Example 1: screening and identification of porcine pseudorabies virus

1. Screening of viral strains

Pig groups injected with a porcine pseudorabies live vaccine (Bartha-K61 strain vaccine) in a disease pig farm in Guizhou in 2018 are subjected to porcine pseudorabies disease, and porcine pseudorabies virus is screened from the disease pig groups. The specific screening steps are as follows:

samples of liver and lymph node of diseased pigs were taken, homogenized with 0.1M PBS solution pH7.2, repeatedly freeze-thawed, centrifuged at 5000r/min for 10min, 10 μl of green streptomycin was added to the supernatant, and the mixture was allowed to stand at 37deg.C for 1h, and filtered through a 0.22 μm filter membrane for sterilization. Adding 10 μl of filtrate into Vero cell (figure 1) which has just grown into monolayer, after lesions appear, removing toxic substances, and repeatedly freezing and thawing at-80deg.C.

2. Detection of viruses

Amplifying PRV gE gene fragments by using primer pairs with the following sequences, amplifying collected virus liquid by using Oligo 7.0 designed primer, and carrying out electrophoresis on PCR amplified products, wherein a target strip is amplified by the virus collected liquid (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 is cultivated and domesticated by Vero cells, and the virus content reaches 10 ^7.0 TCID ₅₀ And/or 0.1ml or more.

3. Virulence detection of viruses

TRZYT strain virus solution (virus content 10) ^7.5 TCID ₅₀ 0.1 ml) was dispensed and the titer was determined at-80℃for 36 months. The virus content was determined to be 10 ^7.1 TCID ₅₀ 0.1ml. The result shows that the strain has better stability.

10 6 week-old BALB/c mice were randomly divided into two groups of 5 mice each, one trial of 200TCID ₅₀ The diluted toxin is injected subcutaneously through the neck, and the inoculation amount is 0.5 mL/dose. The other group was inoculated with an equal amount of PBS as a negative control. Mice were observed and recorded daily for clinical symptoms and mortality. Results mice were vaccinated with the virus solution and exhibited typical pseudorabies symptoms: the ill mice gnaw the snap-in parts, severe skin bleeding, coat shedding, and after 120h inoculation, all the mice inoculated with the virus group die. The control mice had no abnormal changes. Indicating that the strain is a virulent strain.

4. Regression test of animals

The PRV antibody negative piglets of 4-8 days old are inoculated, obvious clinical symptoms appear after 48-72 hours of injection, and the symptoms of unstable gait, reduced milk intake, uncoordinated movement, watered limbs, vomiting or diarrhea of partial pigs and the like appear mainly in the form of body temperature rise. The dissection can find that the meninges are hyperemia, the cerebral spinal fluid is excessive, and the organs such as liver, spleen and the like are necrotized; and can be re-isolated from brain tissue to PRV strains.

5. Antibody neutralization assay

The TRZYT strain and the porcine pseudorabies virus QD strain (herpesvirus I type Porcine herpesvirus Type I, with the preservation number of CGMCC No.10266, which are preserved in China academy of sciences of microorganisms and culture preservation management committee of China, the university of microorganisms, and the national institute of China, the university of microorganisms, and the national area of North Chen West Lu, beijing, are respectively subjected to subculture and purification to prepare inactivated vaccines, mice are respectively immunized, the immunized mice are immunized at an immune dose of 2.0 ml/each, the immunization is performed once every 2 weeks, the total immunization is performed for 3 times, blood collection is performed 2 weeks after the third immunization, serum is separated, and the neutralization experiment is performed after the inactivation at 50 ℃.

And (3) diluting the TRZYT strain virus solution by 100 times, respectively mixing with serum prepared from TRZYT strain and porcine pseudorabies virus QD strain in equal volume, neutralizing for 1 hour at room temperature, inoculating mice, and observing for 96-168 hours after inoculation.

As a result, it was found that mice in the TRZYT strain serum neutralization group of the TRZYT strain had no symptoms, while 30% -40% of mice in the porcine pseudorabies virus QD strain serum neutralization group had porcine pseudorabies symptoms. Namely, the neutralization capacity of the positive serum prepared by taking the strain screened by the application as an antigen on the positive serum is obviously better than that of the positive serum prepared by other strains; the TRZYT virus strain obtained by the method is a novel porcine pseudorabies virus variant strain.

6. Analysis of antigen Gene

The gB, gC, gD, gI genes of the TRZYT strain and the porcine pseudorabies virus QD strain 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 immunity genes of the RZYT strain and the porcine pseudorabies virus QD strain have amino differences, and the TRZYT strain is mutated compared with the previous epidemic strain QD strain (table 1); this is presumably because the antibody serum prepared from QD strain as an antigen has a poor neutralizing effect on TRZYT strain.

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

Example 2: preparation of inactivated vaccine

1. Preparation of virus liquid

Inoculating the strain TRZYT and strain QD into ST cell culture to form a monolayer, rotating at 37deg.C for culturing, collecting the toxic cell culture solution when the lesion reaches 80%, and freezing and thawing for 3 times to obtain virus solution.

2. Inactivation of

A10% formaldehyde solution was added to the harvested virus solution to give a final formaldehyde concentration of 0.1%. Then, the inactivated liquid is put into 2 to 8 ℃ for preservation under the condition of 37 ℃ and 200rpm vibration for inactivation for 24 hours. Taking inactivated virus liquid, horizontally centrifuging at 2000r/min, and filtering with a 0.2um filter membrane filter; ultrafiltering and concentrating the filtered virus liquid with 100KD membrane filter for 5-10 times, wherein the virus content in each ml of virus liquid is not less than 10 ^6.5 TCID50 as a semi-finished antigenic virus solution.

3. Semi-finished antigen testing

1) Sterility testing

And (3) carrying out aseptic inspection according to the annex of the current Chinese veterinary pharmacopoeia to determine the aseptic pollution of the prepared semi-finished product.

2) Inactivation test

Taking the inactivated semi-finished antigen liquid, inoculating the inactivated semi-finished antigen liquid into ST cells according to the ratio of the virus liquid to the growth liquid of 1:10, culturing and observing for 5d at 37 ℃, and carrying out blind transmission for 3 generations on a patient without lesions. As a result, no cytopathy was observed. And preparing the vaccine by the semi-finished antigen after being inspected to be qualified.

4. Preparation of inactivated vaccine

1) Preparing an oil phase:

95 parts of animal white oil and 1 part of aluminum stearate are taken, placed in an oil phase preparation tank, heated to 75 ℃, added with 5 parts of span-80, and maintained for 30 minutes when the heating temperature is 110 ℃, and cooled for standby.

2) Aqueous phase preparation

97 parts of each of the inactivated TRZYT strain and the QD strain semi-finished antigen virus liquid are respectively mixed with 3 parts of tween-80 after sterilization, and the mixture is stirred in a water phase preparation tank until the tween-80 is completely dissolved.

3) Emulsification

2 parts of oil phase is taken and placed in a high-speed shearing machine to be stirred in a slow-speed rotation way, then 1 part of water phase is added, and emulsification is carried out at 10000r/min to finish the preparation of the inactivated vaccine.

Example 3: effect detection of inactivated vaccine

1. Vaccine product inspection

1) Traits (3)

Appearance: the vaccine is milky emulsion;

dosage form: in the form of water-in-oil, a small amount of vaccine is sucked and dripped into cold water, and the vaccine is not diffused except the 1 st drip.

Stability: 5ml of the extracted vaccine is added into a centrifuge tube, and the mixture is centrifuged at 2000r/min for 10 minutes, and the water phase separated out from the bottom of the tube does not exceed 0.2ml.

2) Safety inspection

The BALB/C mice of 6 weeks of age are immunized, the mice have good mental state, normal appetite and no itching symptom after immunization, and the vaccine strain is safe to the mice.

3) Efficacy test

After 10 days of immunization of the mice subjected to safety test, serum was collected and separated, and antibodies were measured. The results showed that the mice in the immunized group remained antibody positive after 100-fold dilution.

2. Toxicity attack protection experiment

The TRZYT strain and the QD strain are prepared into inactivated vaccines according to the method of the example 2, and the commercial porcine pseudorabies virus Bartha-K61 strain vaccine is used for immunizing BALB/C mice of 6 weeks old respectively, wherein the immune dose is 0.5 ml/mouse, and 10 mice are in each group; control groups (injected with PBS buffer) were also included. Immunization was performed once every other week, 3 times in total, 2 weeks after the third immunization, and the TRZYT strain virus solution and the QD strain virus solution collected in example 1 were diluted 10-fold with PBS, respectively, and then subjected to a challenge experiment. The results of the challenge are shown in table 2 below.

Table 2: results of challenge protection test of porcine pseudorabies virus TRZYT strain and QD strain (survival number/total challenge number)

The test result of the toxicity attack protection shows that the TRZYT strain inactivated vaccine can provide complete immune protection for the TRZYT strain, and the group of experimental mice do not show obvious clinical symptoms of porcine pseudorabies and do not have death phenomenon. While providing partial protection against QD strains. The QD strain inactivated vaccine can provide complete protection for the QD strain and only provide partial protection for the TRZYT strain. The Bartha-K61 strain vaccine has poor protection effect on TRZYT strain and QD strain.

The method shows that the cross protection between the TRZYT strain and the QD strain is poor, and also shows that the TRZYT strain is a variant strain of the porcine pseudorabies virus and has obvious difference in virus attack protection from a classical strain, so that the classical strain and the newly screened variant strain are added as vaccine antigens in the vaccine prevention of the porcine pseudorabies in China at present, and the method can play a better role in preventing the porcine pseudorabies virus which is popular at present.

Sequence listing

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

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Pro Glu Tyr Ser Gln Gly Arg Asn Phe Thr Glu Gly Ile Ala Val Leu

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Ile Val Tyr Met Ser Pro Phe Tyr Gly Leu Arg Glu Gly Ala His Gly

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Arg Asn Phe Leu Arg Thr Pro His Phe Thr Val Ala Trp Asp Trp Ala

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Pro Lys Thr Arg Arg Val Cys Ser Leu Ala Lys Trp Arg Glu Ala Glu

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Arg Ala Leu Gly Ala Ser Phe Val Ser Asp Val Thr Gln Leu Asp Leu

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Gly Asp Lys Pro Glu Val Tyr Leu Ala Arg Gly Gly Phe Val Val Ala

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Phe Arg Pro Leu Ile Ser Asn Glu Leu Ala Gln Leu Tyr Ala Arg Glu

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

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

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Pro Pro Ala Val Asn Gly Thr Gly His Leu Arg Ile Thr Thr Gly Ser

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Ala Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asp His Ile Gln Ala His

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Val Asn Asp Met Leu Ser Arg Ile Ala Ala Ala Trp Cys Glu Leu Gln

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Ser Arg Pro Leu Val Thr Phe Glu His Asn Gly Thr Gly Val Ile Glu

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Glu Thr Ile Ser Thr Arg Val Thr Leu Asn Leu Thr Leu Leu Glu Asp

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Thr Gly Leu Leu Asp Tyr Ser Glu Ile Gln Arg Arg Asn Gln Leu His

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Ala Leu Lys Phe Tyr Asp Ile Asp Arg Val Val Lys Val Asp His Asn

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Val Val Leu Leu Arg Gly Ile Ala Asn Phe Phe Gln Gly Leu Gly Asp

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Val Gly Ala Ala Val Gly Lys Val Val Leu Gly Ala Thr Gly Ala Val

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Ile Ser Ala Val Gly Gly Met Val Ser Phe Leu Ser Asn Pro Phe Gly

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Lys Lys Asn Ser Gly Pro Ala Leu Leu Ala Ser Arg Val Gly Ala Met

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Tyr Tyr Arg Arg Ser Ala Leu

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

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

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

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

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

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

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

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

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

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

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

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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

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

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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

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

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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

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

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Gly Pro

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<213> Artificial sequence (Artificial Sequence)

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

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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

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

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

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Claims

1. The porcine pseudorabies virus strain is characterized by being a herpes virus type I porcine pseudorabies virus TRZYT strain, and the preservation number of the porcine pseudorabies virus strain is CGMCC NO.17403.

2. The use of the porcine pseudorabies virus strain of claim 1 in the preparation of a porcine pseudorabies virus attenuated strain.

3. The use of the porcine pseudorabies virus strain of claim 1 in the preparation of an inactivated vaccine.