CN107267470A - A kind of gE plants of PRV gene-deleted strain C1201/ Δs and its application - Google Patents

Abstract

The invention provides a porcine pseudorabies virus gene deletion strain C1201/ΔgE strain, the porcine pseudorabies virus (Pseudorabies Virus, PRV) is a gE gene deletion strain, and its microbial preservation name is: porcine pseudorabies virus gene deletion strain C1201/ΔgE strain; preservation number: CCTCC NO: V201721; preservation unit: China Center for Typical Culture Collection; preservation address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, preservation date: 20170504, the porcine pseudorabies virus C1201/ The ΔgE strain is continuously passaged on cells, has good stability and does not mutate, and provides a good resource for the development of porcine pseudorabies gene-deleted inactivated vaccines.

Description

A kind of porcine pseudorabies virus gene deletion strain C1201/ΔgE strain and its application

technical field

The invention relates to the field of production of virus live vaccines for animals, in particular to a porcine pseudorabies virus gene deletion strain C1201/ΔgE strain and application thereof.

Background technique

Porcine herpesvirus type I (Suid herpesvirus I), also known as pseudorabies virus (Pseudorabies virus, PRV), can cause a variety of domestic animals, experimental animals and wild animals with high fever, severe itching and neurological symptoms of varying degrees (Jakubik, 1977). Pigs are the only natural host of PRV, and all age groups can be infected. Infected piglets often develop neurological disorders (such as vomiting, tremors, ataxia, paralysis, and convulsions) and may die from severe encephalomyelitis; adult pigs mainly present with fever and respiratory symptoms (sneezing and pneumonia); pregnancy Sow infection can lead to miscarriage or stillbirth, and pigs of any age can develop latent infection after being resistant to acute infection (Mettenleiter, 2000). Therefore, this disease has caused huge economic losses to the pig industry in many countries, and has aroused great attention of veterinary workers in various countries in the world.

The disease has been eradicated from domestic pig herds in many European countries through increased control and eradication programs (culling of wild virus-infected pigs on the basis of mass vaccination against gE deficiency to differentiate wild-virus infection from vaccination) , these countries include Austria, Denmark, Finland, Germany, the Netherlands, Sweden, Switzerland, Norway, etc. Elimination of the disease in domestic pigs has also been reported in Canada, New Zealand and the United States in recent years.

The prevention and control of this disease in my country mainly adopts immunization with gE gene deletion vaccine combined with gE-ELISA detection method, but pseudorabies still often occurs. Since 2011, there have been suspected pseudorabies epidemics in large-scale pig farms in many provinces in my country, such as abortion, stillbirth, and death of newborn piglets after neurological symptoms. After investigation, these pig farms have been immunized with pseudorabies vaccine, which may be caused by an antigenically mutated pseudorabies virus (Yang Hanchun, 2013). We collected suspected porcine pseudorabies disease material from PRV vaccine immunized pig farms and inoculated PK-15 cells, and isolated a strain of PRV, which was named BJ-11 strain. The results of whole genome sequence analysis showed that the isolated strain was a new epidemic strain. The BJ-11 strain has strong pathogenicity to immunized piglets, with a lethal rate of 40%. The existing Bartha K61 vaccine can no longer provide complete immune protection for piglets.

Contents of the invention

In order to overcome the defects in the above-mentioned prior art, the present invention provides a porcine pseudorabies gene deletion strain (Pseudorabies Virus, PRV) C1201/ΔgE strain, its microbial preservation number is: CCTCC NO: V201721; preservation time: 2017.05.04; Deposit unit: China Center for Type Culture Collection, CHINA CENTER FOR TYPE CULTURE COLLECTION (CCTCC); deposit address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province.

The above-mentioned porcine pseudorabies gene deletion strain, wherein, the US8 gene of the gE protein of the virus strain is completely deleted, and the US7 gene of the gI protein is missing 872-1101 bases, that is to say, the porcine pseudorabies virus C1201/ΔgE strain The gene sequence is missing all of gE and part of gI, and the US7 gene of gI protein is missing 872-1101 bases.

The present invention may also include: the application of a porcine pseudorabies virus gene deletion strain C1201/ΔgE strain, which can be applied to the immunity of weaned piglets to new epidemic strains, and the protection rate is not lower than 80%. Immunization of weaned piglets using C1201/ΔgE prepared inactivated vaccine can resist the challenge of the newly popular and virulent HB-1201 strain of porcine pseudorabies.

The present invention has the following advantages:

1. The C1201/ΔgE strain of porcine pseudorabies virus constructed by the present invention is continuously passaged on cells, has good stability and does not mutate, and provides good resources for the development of porcine pseudorabies gene-deleted inactivated vaccines.

Description of drawings

The invention and its characteristics, shapes and advantages will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings. Like numbers designate like parts throughout the drawings. The drawings are not intended to be drawn to scale, emphasis instead being placed upon illustrating the gist of the invention.

Fig. 1 is the construction process of PRV C1201/ΔgE of the present invention.

Fig. 2 is the gene sequence of the PRV C1201/ΔgE deletion part of the present invention.

Fig. 3 is a schematic diagram of the construction of the transfer vector in the present invention.

Fig. 4a and Fig. 4b are schematic diagrams of fluorescence observation and cytopathic changes after inoculation of the purified recombinant virus in the present invention into PK-15 cells.

Figure 5a is a schematic diagram of the CPE results of rPRV-GFP-gE-on Vero cells, Figure 5b is a schematic diagram of the IFA results of rPRV-GFP-gE-on Vero cells, and Figure 5c is the CPE results of PRV C1201/ΔgE on Vero cells Schematic diagram, Figure 5d is a schematic diagram of IFA results of PRV C1201/ΔgE on Vero cells.

Fig. 6 is a schematic diagram of the results of the immune challenge test of weaned piglets according to the embodiment of the present invention.

Fig. 7 is a schematic diagram of body temperature recording of weaned piglets after immune challenge according to the embodiment of the present invention.

8a-8f are schematic diagrams of autopsy results of WN16 in the challenge control group and WN12 in the immunized group in the embodiment of the present invention.

Figures 9a-9d are schematic diagrams of IHC results of the brain and spleen of WN16 in the challenge control group and WN12 in the immune group in the embodiment of the present invention.

Figure 10 is the electrophoresis diagram of F1 generation C1201/ΔgE.

Fig. 11 is the electrophoresis diagram of F1, F2, F7, F12, F16 generation C1201/ΔgE.

Fig. 12 is a peak map of F0 generation C1201/ΔgE forward sequencing.

Fig. 13 is a peak map of reverse sequencing of F0 generation C1201/ΔgE.

Figure 14 shows the genetic distance results of forward sequencing.

Figure 15 shows the sequence alignment results of forward sequencing.

Figure 16 is the genetic distance result of reverse sequencing.

Figure 17 is the sequence alignment result of reverse sequencing.

detailed description

In the following description, numerous specific details are given in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without one or more of these details. In other examples, some technical features known in the art are not described in order to avoid confusion with the present invention.

In order to thoroughly understand the present invention, detailed steps and detailed structures will be provided in the following description, so as to illustrate the technical solution of the present invention. Preferred embodiments of the present invention are described in detail below, however, the present invention may have other embodiments besides these detailed descriptions.

With reference to Fig. 1-shown in Fig. 17, the present invention provides a kind of porcine pseudorabies virus gene deletion strain C1201/ΔgE strain, porcine pseudorabies virus (Pseudorabies Virus, PRV) is the gE gene deletion strain, and its microbial preservation name is: porcine pseudorabies virus Rabies virus gene deletion strain C1201/ΔgE strain; preservation number: CCTCC NO:V201721; preservation unit: China Center for Type Culture Collection; preservation address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, preservation date: 20170504, further, The US8 gene of the gE protein of the virus strain is completely deleted, and the US7 gene of the gI protein is missing 872-1101 bases.

Example 1

(1) Construction of gE gene transfer vector

Using the PRV strain with GenBank accession number BK001744.1 and the sequence of pEGFP-N1 as a reference template, design primers to amplify the PRV gE gene, PRV gC gene and EGFP gene, apply the fusion PCR method, and construct the transfer vector according to the strategy shown in Figure 3 , in Figure 3, A represents the region of the fusion fragment; B represents the loxP site.

With KOD FX DNA Polymerase was used to amplify the target fragment, and a 50 μL reaction system was constructed. The amplification conditions were: 94°C for 10 minutes; 98°C for 20 seconds, 56-66°C for 45 seconds, 68°C at 1 kb/min for a total of 38 cycles; 68°C for 7 minutes. 1) Extract the PRVC1201/ΔGE genome, use the extracted genome as a template, use primers gE-L F/R, gE-R F/R to amplify the homology arms gE-L, gE- R, wherein a fusion fragment of 13 bp is added to the end of gE-L and the beginning of gE-R. After the amplified products were checked by 1% agarose gel electrophoresis, the SV GEl and PCR Clean-Up System Kit for purification and recovery. The purified and recovered PCR product was ligated into the pJET1.2/blunt Cloning Vector vector, and the ligated product was transformed into Trans10 competent cells. PCR was performed with pJET1.2F/R as primers to identify monoclonal bacteria and perform sequence determination. Plasmids were extracted using the Pure Yield Plasmid Midiprep System kit, and the obtained recombinant plasmids were identified by PCR and enzyme digestion, and the concentration was measured with a UV spectrophotometer, and stored at -20°C. 2) Using pEGFP-N1 as a template and EGFPF/R as a primer to amplify the EGFP expression cassette. After the amplified product was purified and recovered, it was connected with pJET1.2/blunt Cloning Vector, transformed into Trans10 competent cells, and the sequence of the monoclonal bacteria identified as positive by PCR was determined. Extract the recombinant plasmid, measure the concentration with a UV spectrophotometer after identification by PCR and enzyme digestion, and store it at -20°C. 3) First, use the gE-L and EGFP recombinant plasmids verified by sequence determination as templates, and gE-L F and EGFP R as primers, perform fusion PCR to amplify and obtain the fusion fragment gE-L-EGFP. The PCR reaction conditions were: 94°C for 5 min; 11 cycles at 98°C for 10 s, 63°C for 45 s, and 68°C for 1 min; 26 cycles were continued after adding primers and templates; 68°C for 7 min. Secondly, the gE-R recombinant plasmid verified by sequence determination and the fusion fragment gE-L-EGFP after purification and recovery were used as templates, and gE-L F and gE-R R were used as primers to perform fusion PCR to amplify the fusion fragment gE -L-EGFP-gE-R. After the fusion fragment gE-L-EGFP-gE-R was purified and recovered, it was ligated into pJET1.2/bluntCloning Vector and transformed into Trans10 competent cells. The monoclonal bacteria identified as positive by PCR were sequenced. Extract the recombinant plasmid of the transfer vector, measure the concentration with a UV spectrophotometer after identification by PCR and enzyme digestion, and store it at -20°C.

(2) Screening and purification of recombinant virus

Co-transfection: Use the Pure Yield Plasmid Midiprep System kit to extract the transfer vector recombinant plasmid. Select PK-15 cells with normal morphology and vigorous growth, use promega MammalianTransfection System was used for transfection. The specific operation method is as follows: add 20 μg PRV genome, 10 μg transfer vector, 62 μL 2M CaCl2 to tube 1, and finally make up to 500 μL with sterilized water; add 2×PBS 500 μL to tube 2; Add the liquid into tube 2 drop by drop and mix well; incubate the mixed liquid at room temperature for 30 minutes; shake and mix again, immediately add dropwise to the cell culture dish, mix well, and place the cell culture dish at 37°C in 5% CO2 cultured in an incubator.

Screening of recombinant virus and plaque purification: When cell lesions with positive fluorescent signals appear, freeze and thaw the cell culture medium repeatedly, centrifuge at 4500r/min at 4°C for 10min, and aspirate the supernatant. The supernatant was diluted 10-fold with serum-free and antibiotic-free DMEM medium. Take 1 mL of the virus solution with a dilution ratio of 10-2-10-7 and inoculate a well-grown PK-15 cell monolayer in a 6-well cell culture plate, and act for 1 hour at 37°C and 5% CO2. Wash 3 times with serum-free and antibiotic-free DMEM medium. Heat and melt 2% low-melting point agarose, and place it in a 37°C water bath to cool down. At the same time, put 20% 2×DMEM medium in a 37°C water bath, and mix at a ratio of 1:1 after the temperature is consistent. Add 2 mL of mixed agarose gel to each well and place it at 4°C for 10 min. After the agarose gel is solidified, transfer the cell culture plate to a 37°C CO2 cell incubator for culture. Observe and mark a single plaque with a green fluorescent signal under an inverted fluorescent microscope, absorb the plaque showing a green fluorescent signal at the maximum dilution factor, pipette it into 1 mL of serum-free and antibiotic-free DMEM nutrient solution, and repeat freeze-thaw. The above operations were repeated, and the obtained plaque fluid was subjected to several rounds of plaque purification. The obtained recombinant virus was named rPRV-GFP-gE-. Referring to Figure 4a and Figure 4b, the CPE produced by the recombinant virus on PK-15 cells and the green fluorescent signal observed under the fluorescence microscope. It can be seen from Figure 4a that the recombinant virus has entered the PK-15 cells, indicating that it is active .

(3) Obtaining and purifying PRV gE gene deletion strain C1201/ΔgE

Extract the genome of the recombinant virus rPRV-GFP-gE-, and perform Cre recombinase treatment according to the following system: 2 μL 10×Cre Buffer, 10 μg target DNA, 5 μL Cre enzyme (1UI/μL), and supplement ultrapure water to 30 μL. Mix well, place in 37°C water bath for 1 hour, and then place at 70°C for 10 minutes.

Extract the viral DNA after the Cre enzyme treatment, the specific operation method is as follows: add 200 μ L of phenol: chloroform: isoamyl alcohol (25:24:1) to the reaction system after the Cre enzyme treatment, extract twice, and take the supernatant after centrifugation; Add 500 μL of pre-cooled absolute ethanol, place at -20°C for precipitation for 10 minutes, centrifuge at 12,000 rpm/min for 15 minutes, discard the supernatant; add 70% ethanol to wash the precipitate and dry it in an incubator. The resulting precipitate was dissolved in an appropriate amount of TE solution, its concentration was measured with an ultraviolet spectrophotometer, and stored at -20°C.

Add 500 μL Opti-MEM, 6 μL Cre-treated viral DNA and 6 μL PLUS to the sterilized EP tube, mix well and let stand for 5 minutes; then add 12 μL LTX, mix evenly and let stand for 30 minutes. Select vigorously growing Vero cells. All the mixed solution was added to the cell culture solution, and cultured in a 5% CO2 incubator at 37°C. After 12 hours, the 5% DMEM culture medium was replaced. Observe the CPE, and observe the green fluorescence generation under a fluorescence microscope, and mark the area where the CPE is obvious but no obvious fluorescent signal is observed. Aspirate the cells in the selected area and put them in 5% DMEM medium. The obtained virus liquid was subjected to plaque purification, and the obtained gene deletion strain was named PRV C1201/ΔgE. 5a-5d show the CPE and indirect immunofluorescence detection results of the purified PRV C1201/ΔgE on Vero cells.

(4) Sequence determination of PRV C1201/ΔgE

The genome of PRV C1201/ΔgE was extracted and used as a template, and gE-LF and gE-RR were used as primers for PCR amplification. After the PCR product was purified and recovered, it was ligated into pJET1.2/blunt Cloning Vector and transformed into Trans10 competent cells. The monoclonal bacteria identified as positive by PCR were sequenced. Sequencing results showed that, compared with the parental strain, the gene deletion region of PRVC1201/ΔgE included bases 872-1101 of the US7 gene encoding gI protein, the Gap region between US7 and US8, and the entire US8 gene encoding gE protein.

2. Genetic stability test:

Cells were inoculated, and the strain obtained by cloning and purification was the primary generation, that is, the P0 generation. The gene changes of the 6 generations were compared by sequencing with P0.

The results showed that there were no significant differences in clinical symptoms and pathological autopsies among the six generations of viruses. The results of clinical animal experiments showed that the C1201/ΔgE strain continuously returned to animals, and the toxicity did not return to strong.

Similar to the classic natural attenuated strain Bartha-k61, the pseudorabies virus gene deletion strain C1201/ΔgE has a large number of nucleotide deletions, and the deletion region includes part of gI and all gE, specifically the 872-1101 base of the US7 gene encoding the gI protein base, the Gap region between US7 and US8, and the entire US8 gene encoding the gE protein. After the gene deletion strain was passed in pigs for 5 times, the target sequence was amplified without any sequence insertion.

In order to further verify whether the vaccine virus of the 5 generations has mutated, the amplified PCR products were gel-cut and recovered, and the PCR products were directly sent to the sequencing company for sequencing in both positive and negative directions. The results showed that the sequence homology between the P0 virus and other generations was 99.9-100% in the forward direction, and 99.8-100% in the reverse direction. The results of sequence comparison of the corresponding positions between the gene deletion strain C1201/ΔgE of the P0 generation and the Bartha-K61 strain showed that the similarity of the 3' end was 56.6%, while that of the 5' end was only 54.5%, indicating that C1201/ΔgE and the Bartha-K61 strain Not the same strain. The peak diagrams of the forward and reverse sequencing of the P0 generation virus are shown in Figure 12 and Figure 13, the genetic distance results of the forward sequencing are shown in Figure 14, the sequence comparison results of the forward sequencing are shown in Figure 15, and the results of the reverse sequencing are shown in Figure 15. The result of genetic distance is shown in FIG. 16 , and the sequence alignment result of reverse sequencing is shown in FIG. 17 .

3. Inactivated immune potency test

3.1 Grouping: The weaned piglets used in the experiment were randomly divided into 3 immunization groups (5 heads in each group), and a challenge control group (5 heads) and a blank control group (5 heads) were also set up.

3.2 Preparation of inactivated vaccine: adjust the virus content before inactivation to 107.5TCID50/mL, and prepare with immune adjuvant 5:1 to immunize piglets.

3.3 Immunization: The prepared inactivated vaccine was immunized in groups according to the intramuscular injection of 2ml of vaccine into each pig's neck.

3.5 Challenge: 28 days after immunization, 3 immunization groups and the challenge control group were challenged with PRV BJ-11 strain virulence (104.0 TCID50/mL). At the same time nasal drops 3.0ml. The blank control group was not challenged.

3.6 Observation after challenge: The piglets were observed continuously for 21 days after challenge, and their body temperature was measured every day to observe the appetite, mental state, dyspnea, diarrhea, runny nose, death and clinical symptoms of the piglets.

3.8 Judgment criteria

3.8.1 Judgment criteria for the onset of the disease after the attack: the occurrence of the following item (3), or the occurrence of the conditions (1) and (2) at the same time will be judged as the onset.

(1) Depression, loss of appetite or dyspnea (including coughing, panting, mouth breathing, abdominal breathing), runny nose, and diarrhea;

(2) Body temperature ≥ 40.5°C for at least 3 days;

(3) Obvious neurological symptoms (ataxia, paralysis, scrambling of limbs, opisthotonus, etc.) or death.

4. Results

4.1 Immune effect of the vaccine on weaned piglets After 28 days of challenge after immunization and 21 days of continuous observation, the protection rate of the three batches of vaccine immunization groups was greater than 80%. The morbidity rate of piglets in the non-immunized challenge group was 5/5 (100%), and neurological symptoms such as ataxia, paralysis, and scrambling of limbs occurred in 2 pigs; another pig died. The blank control group had no adverse reactions. The test results are shown in Figure 6, and the temperature measurement records are shown in Figure 7.

Referring to Figure 8a-Figure 8f, Figure 9a-Figure 9d, wherein Figure 8a, Figure 8c, Figure 8e are the challenge group of lung, spleen, and tonsil respectively, Figure 8b, Figure 8d, Figure 8f are the immune group, Figure 9a , Figure 9c are the brain IHC of the challenge group and the spleen IHC of the challenge group respectively, Figures 9b and 9d are the brain IHC of the immune group and the spleen IHC of the immune group respectively, the autopsy results are: after the pigs in the challenge control group were dissected, the lungs There were severe gray-white necrotic nodules, hemorrhage, and edema, necrotic foci in the spleen, hemorrhage in the tonsils, and IHC results of the brain and spleen were positive. There were no obvious pathological changes in lung, spleen and tonsil of other immunized pigs.

5 Conclusion

The test results show that the protection rate of the vaccine against new epidemic strains after immunizing weaned piglets is not less than 80%. Immunization of weaned piglets using C1201/ΔgE prepared inactivated vaccine can resist the challenge of the newly popular and virulent HB-1201 strain of porcine pseudorabies.

The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; Under the circumstances of the technical solution of the invention, many possible changes and modifications can be made to the technical solution of the present invention by using the methods and technical contents disclosed above, or be modified into equivalent embodiments with equivalent changes, which does not affect the essence of the present invention . Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention, which do not deviate from the technical solution of the present invention, still fall within the protection scope of the technical solution of the present invention.

sequence listing

<110> Shanghai Chuanghong Biotechnology Co., Ltd.

<120> A Gene Deletion Strain of Porcine Pseudorabies Virus C1201/ΔgE Strain and Its Application

<210> 1

<211> 2081

<212>DNA

<213> Pseudorabies Virus

<400> 1

tcgttgccgc gcccgcctcg gcgccggcgc acgtcgcccc gggtcacgat ctgggagtgc 60

agggcctccg tccactcgcc ggcgtggcgc caggagcggt tgtggacccg cgcgaacatg 120

gcgcgggtcg cctggatccc ggcgacgatc acgtccaggg cgtcggcgtc cgtcagcccg 180

ggccggcgcc gggtcaggcg cgcgcccgtc tgggcgtggg aggctccggc ggcggtgctg 240

cgggaggcgg ccaggagcac ctggtcgcag aggtcggcgg cgcccaggat ccacaggtgg 300

acgggggccg tgccccgggc ccccgagttc aggtactgga tcccgttcag gacggacgac 360

cactccgtgt ccagccgcgg gggtgggctg atcctgaggg gctccccggg cttcgagccg 420

tccgccgggg ggcgccgcgt cagctcgtgc gtctcggtgg tgatgtagaa cggcgccgtg 480

gggtcgggacc gcgtccgcac gacggggcgc acggcgcgcg gcagcagggc cagcgagccg 540

ggggagatct ccgaggagcg cagcaccacg tgctgccgcg gcgagttctc gtcggcggcg 600

cgccgctgct gcaccatcga cgcgtagcac cactcggtga gcaccttcca caggtccggg 660

tgcgcctcgc ccacgaaggc ggggatgcgc ccggccccgt ccatcagcgt gaccacggtg 720

atggccgtca cccccatggt gcgaacgcgc tcgcgacaac tgcgacggtg gaggcggtgg 780

agaagaagag tccggcgaga gacgggcgga acagagacgc ggaggagagg acggctgctg 840

tgtgcgcccg gacacgccgg ggcccattta ttgtgacaag tccgagtccg tgcccacacc 900

acgtggcgcg cccaaccccc cgctctctcc cccctctcct gggcgcggcg gatgggggcg 960

ggcccccgct cccggtcgct cgctcgctcg ctacacgtgc ctggcgacga tgcccccgag 1020

tagcgcggac agcgagcaga tgaccagcgc ggcggcgctg atcgcgacgc ccatcaggca 1080

gcggcggcgt ctccgacgcg ccgcctgccg gcgtcccacg cggcgcagga actcgctggg 1140

cgtctcgttg tcgctctcgc tgtagtagca gtccgagtcg tcctgggggc gcagcgggga 1200

gcggggtccc ttgggggcca gcaggacgtc ggcggccggg ttcgagacgc tcgtcgggac 1260

gggggcgctg gggtcaaacg tgtccatgtc gacggaggcg gcgccgggca tgtcggaatg 1320

cgggcggacc ggttctcccg gtatataact tcgtatagca tacattatac gaagttatcc 1380

agcaccatgg ctatcttccc ggggctccgg gcgcccgccg tggcgttggc ggcggcgagc 1440

aggacgcgcg acacgacgct ggcgttcagc accatcgtgc cgttcgcgtc cagggtcccg 1500

ggcgccgggg tcagcgtcgg cgtcgtcgtc tccgcgtcac cctcctcctc ctcgtccgat 1560

cggggctcgg gggtggcgcc ggtcccccgg gggggcgcgg gggtcgtcgg cggctcgggc 1620

tcgggcgggg gctgggtggt gaacacggcg tcctcggcgg ggtccacgac gcgcaggctg 1680

tcgtgccacg atccgacgac gggccggcac tcgtccgcgg gcggcgatgt ggggacgggg 1740

cgccccctcg gcggcaccag ggccgtcagc acaaagaggt ccgtggtccc gttcacgcgg 1800

acccgcagca cgtacgaccc cgcgtccccc gaggccgggc gcgagacgaa cagcagccgg 1860

cgcgcctcca ccgcggcgga cgcgcgccgg gcgagcggct cgcgcttgcg caggcagccg 1920

cggaaggctt cgtggtgcac gcggggggca gaggtcgtac tcggcggcgt actcgcgcgt 1980

gtagcaggcg cgcttggggt cgaggcgcag cagctccacg cgcccgctgt agttgctcgg 2040

cgagggcccc tccagaaaca gcagcgtccc gtctatcgtc a 2081

Claims (2)

1. a kind of gE plants of PRV gene-deleted strain C1201/ Δs, it is characterised in that PRV (Pseudorabies Virus, PRV) is gE gene-deleted strains, and its microbial preservation is entitled：Porcine pseudorabies virus gene lacks Lose gE plants of strain C1201/ Δs；Preserving number is：CCTCC NO:V201721；Depositary institution：China typical culture collection center；Protect Hide address：Wuhan City, Hubei Province Wuchang District Wuhan University collection, preservation date：20170504.

2. a kind of gE plants of PRV gene-deleted strain C1201/ Δs according to claim 1, it is characterised in that should The US8 genes of Strain gE albumen are lacked completely, and gI albumen US7 gene delection 872-1101 bit bases.