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

Abstract

The invention provides a kind of gE plants of PRV gene-deleted strain C1201/ Δs, the PRV (Pseudorabies Virus, PRV) is gE gene-deleted strains, and its microbial preservation is entitled：GE plants of PRV gene-deleted strain C1201/ Δs；Preserving number is：CCTCC NO:V201721；Depositary institution：China typical culture collection center；Preservation address：Wuhan City, Hubei Province Wuchang District Wuhan University collection, preservation date：20170504, the continuous passage on cell for gE plants of the PRV C1201/ Δs that the present invention is built with good stability, is not morphed, the development for pseudorabies gene delection inactivated vaccine provides good resource.

Description

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

Technical field

Field is manufactured with viral lived vaccine, and in particular to the present invention relates to animal to a kind of PRV gene delection GE plants of C1201/ Δs of strain and its application.

Background technology

Herpesvirus suis I types (Suid herpesvirus I) be also known as pseudorabies virus (Pseudorabies virus, PRV) can cause a variety of domestic animals, experimental animal and wild animal hyperpyrexia, it is strange itch and in various degree nervous symptoms (Jakubik, 1977).Pig is the unique natural reservoir (of bird flu viruses) of PRV, and each age group can infect.The nervous system disease is often developed into after Infection in Piglets (as vomitted, trembling, incoordination is paralysed and twitched), and serious encephalomyelitis may be died from；Adult Pig mainly generates heat (sneezed and pneumonia) with respiratory symptom；It can cause to miscarry or produce stillborn foetus after farrowing sow infection, the pig at any age is resistance to Latent infection (Mettenleiter, 2000) can be formed by crossing after acute infection.Therefore, this disease is made to many national pig industrys Into huge economic loss, the great attention of countries in the world veterinarian is caused.

By increase control dynamics and implement elimination plan (in large-scale inoculation gE deletion of vaccine with distinguish wild virus infection with The pig of wild virus infection is slaughtered on the basis of vaccine inoculation), many countries in Europe eradicate this disease in domestic pig group, these Country includes Austria, Denmark, Finland, Germany, Holland, Sweden, Switzerland, Norway etc..In recent years, Canada, New Zealand and U.S. State, which has also been reported in domestic pig, eliminates this disease.

China mainly uses immunity inoculation gE gene-deleted vaccine combination gE-ELISA detection methods to this sick prevention and control, but Pseudoabies still often has generation.Since 2011, there is miscarriage, production stillborn foetus, first cub in many province large-scale pig farm sows of China There is the epidemic situation of the doubtful pseudoabies such as death after nervous symptoms in pig, and indivedual provinces are more serious, and morbid pig number is more, loss It is larger.The immunity inoculation of pseudorabies vaccines was carried out through investigating these pig farms, this phenomenon is probably by a kind of antigen Caused by the pseudorabies virus that property morphs (Yang Hanchun, 2013).It is pseudo- that we gather doubtful pig from PRV vaccine immunities pig farm Rabies pathological material of disease is inoculated with PK-15 cells, and isolated 1 plant of PRV is named as BJ-11 plants.Complete genome sequencing result shows Show, the isolated strain is new popular strain.BJ-11 plants have stronger pathogenic to piglet is immunized, and fatal rate is existing up to 40% Bartha K61 vaccines complete immunoprotection can not be provided for piglet.

The content of the invention

The present invention provides a kind of pseudorabies gene delection strain to overcome the defect of above-mentioned prior art presence (Pseudorabies Virus, PRV) gE plants of C1201/ Δs, its microbial preservation number is：CCTCC NO:V201721；During preservation Between：2017.05.04；Depositary institution：China typical culture collection center, CHINA CENTER FOR TYPE CULTURE COLLECTION(CCTCC)；Preservation address：Wuhan City, Hubei Province Wuchang District Wuhan University collection.

Above-mentioned pseudorabies gene delection strain, wherein, the US8 genes of Strain gE albumen are lacked completely, and The US7 gene delection 872-1101 bit bases of gI albumen, that is to say, that the gene order of gE plants of PRV C1201/ Δs Whole gE and partial gI are lacked, wherein, the US7 gene delection 872-1101 bit bases of gI albumen.

The present invention can also include：A kind of application of gE plants of PRV gene-deleted strain C1201/ Δs, can be applied New popular strain is immunized in weanling pig, protective rate is not less than 80%.Weanling pig is immune to be prepared using C1201/ Δs gE Inactivated vaccine can resist the attack of new popular strong HB-1201 plants of the poison of pseudorabies.

The present invention has advantages below：

1st, the continuous passage on cell for gE plants of the PRV C1201/ Δs that the present invention is built, with steady well It is qualitative, do not morph, the development for pseudorabies gene delection inactivated vaccine provides good resource.

Brief description of the drawings

By reading the detailed description made with reference to the following drawings to non-limiting example, the present invention and its feature, outside Shape and advantage will become more apparent upon.Identical mark indicates identical part in whole accompanying drawings.Not deliberately proportionally Draw accompanying drawing, it is preferred that emphasis is the purport of the present invention is shown.

Fig. 1 is that PRV C1201/ Δs gE of the present invention builds flow.

Fig. 2 is the gene order of PRV C1201/ Δs gE lack parts of the present invention.

Fig. 3 builds schematic diagram for transfer vector in the present invention.

Fig. 4 a, Fig. 4 b are inoculated with Fluirescence observation and its cytopathy after PK-15 cells for the recombinant virus purified in the present invention Become schematic diagram.

Fig. 5 a are that CPE result schematic diagrams of the rPRV-GFP-gE- on Vero cells, Fig. 5 b are that rPRV-GFP-gE- exists CPE result schematic diagrams that IFA result schematic diagrams, Fig. 5 c on Vero cells are PRV C1201/ Δs gE on Vero cells, figure 5d is IFA result schematic diagrams of the PRV C1201/ Δs gE on Vero cells.

Fig. 6 is wean piglet immunological challenge test result schematic diagram in the embodiment of the present invention.

The temperature recording schematic diagram that Fig. 7 is attacked after poison for the piglet immunological that weans in the embodiment of the present invention.

Fig. 8 a- Fig. 8 f be the embodiment of the present invention in attack malicious control group WN16 and immune group WN12 cut open inspection result schematic diagrams.

Fig. 9 a- Fig. 9 d illustrate to attack malicious control group WN16 and immune group WN12 brains, spleen IHC results in the embodiment of the present invention Figure.

Figure 10 is F1 generation C1201/ Δ gE electrophoretograms.

Figure 11 is F1, F2, F7, F12, F16 for C1201/ Δ gE electrophoretograms.

Figure 12 is peak figures of the F0 for C1201/ Δs gE forward direction sequencings.

Figure 13 is peak figures of the F0 for C1201/ Δ gE backward sequencings.

Figure 14 is the positive genetic distance result being sequenced.

Figure 15 is the positive sequence alignment result being sequenced.

Figure 16 is the genetic distance result of backward sequencing.

Figure 17 is the sequence alignment result of backward sequencing.

Embodiment

In the following description, a large amount of concrete details are given to provide more thorough understanding of the invention.So And, it is obvious to the skilled person that the present invention can be able to without one or more of these details Implement.In other examples, in order to avoid obscuring with the present invention, do not enter for some technical characteristics well known in the art Row description.

In order to thoroughly understand the present invention, detailed step and detailed structure will be proposed in following description, so as to Explain technical scheme.Presently preferred embodiments of the present invention is described in detail as follows, but in addition to these detailed descriptions, this Invention can also have other embodiment.

Shown in reference picture 1- Figure 17, the invention provides a kind of gE plants of PRV gene-deleted strain C1201/ Δs, PRV (Pseudorabies Virus, PRV) is gE gene-deleted strains, and its microbial preservation is entitled：Pig puppet is mad GE plants of dog disease virus gene gene-deleted strain C1201/ Δs；Preserving number is：CCTCC NO:V201721；Depositary institution：Chinese Typical Representative culture Thing collection；Preservation address：Wuhan City, Hubei Province Wuchang District Wuhan University collection, preservation date：20170504, enter one Step, the US8 genes of Strain gE albumen lack completely, and gI albumen US7 gene delection 872-1101 bit bases.

Embodiment 1

(1) structure of gE gene transfer vectors

PRV strains and pEGFP-N1 sequences by BK001744.1 of GenBank indexed numbers are reference template, design primer PRV gE genes, PRV gC genes and EGFP gene are expanded, using fusion DNA vaccine method, shifts and carries according to construction of strategy shown in Fig. 3 Body, in figure 3, A represent to merge segment area；B represents loxP sites.

Use KOD FXDNA Polymerase expand purpose fragment, build 50 μ L reaction systems, amplification condition For：94℃10min；98 DEG C of 20s, 56-66 DEG C of 45s, 68 DEG C of 1kb/min totally 38 circulations；68℃7min.1) PRV is extracted C1201/ Δ GE genomes, using the genome of extraction as template, US8 (gE) is expanded with primer gE-L F/R, gE-R F/R respectively Homology arm gE-L, gE-R of gene left and right ends, wherein gE-L ends and gE-R initiating terminals respectively add 13bp fusion fragment. After amplified production is checked through 1% agarose gel electrophoresis, useSV GEl and PCR Clean-Up System are tried Agent box carries out purifying recovery.PCR primer after purifying is reclaimed is connected in pJET1.2/blunt Cloning Vector carriers, Connection product is transformed into Trans10 competent cells, and performing PCR is entered by primer of pJET1.2F/R, identifies monoclonal bacterium, carries out sequence Row are determined.Plasmid extraction uses Pure Yield Plasmid Midiprep System kits, and the recombinant plasmid of acquisition is passed through After PCR and digestion are identified, concentration is determined with ultraviolet specrophotometer, -20 DEG C of preservations are put.2) using pEGFP-N1 as template, EGFP F/R is primer, expands EGFP expression cassettes.Amplified production purifying connects after reclaiming with pJET1.2/blunt Cloning Vector Connect, convert after Trans10 competent cells, the monoclonal bacterium that PCR is accredited as the positive carries out sequencing.Extract recombinant plasmid, Concentration is determined with ultraviolet specrophotometer after PCR and digestion identification, -20 DEG C of preservations are put.3) it is first, correct with sequencing checking GE-L, EGFP recombinant plasmid be template, gE-L F and EGFP R be primer, carry out fusion DNA vaccine, amplification obtain fusion fragment gE-L-EGFP.PCR reaction conditions are：94℃5min；98 DEG C of 10s, 63 DEG C of 45s, 68 DEG C of 1min totally 11 circulations；Add primer And proceed 26 circulations after template；68℃7min.Secondly, correct gE-R recombinant plasmids and purifying are verified with sequencing Fusion fragment gE-L-EGFP after recovery is as template, and gE-L F and gE-R R are primer, carries out fusion DNA vaccine, and amplification is melted Close fragment gE-L-EGFP-gE-R.Fusion fragment gE-L-EGFP-gE-R is purified after recovery, connected into pJET1.2/blunt Cloning Vector simultaneously convert Trans10 competent cells, and the monoclonal bacterium that PCR is accredited as the positive carries out sequencing.Carry Transfer vector recombinant plasmid is taken, concentration is determined with ultraviolet specrophotometer after PCR and digestion identification, puts -20 DEG C of preservations.

(2) screening and purifying of recombinant virus

Cotransfection：Transfer vector restructuring matter is carried out using Pure Yield Plasmid Midiprep System kits The extraction of grain.Selection form is normal, eugonic PK-15 cells, uses promega Mammalian Transfection System are transfected.Concrete operation method is as follows：20 μ g PRV genomes, 10 μ g are separately added into pipe 1 Transfer vector, 62 μ L 2M CaCl2, is finally supplemented to 500 μ L with aqua sterilisa；The μ L of 2 × PBS 500 are added in pipe 2；Jiggle Pipe 2, the liquid in pipe 1 is added dropwise in pipe 2 and fully mixed；Mixing liquid is incubated 30 minutes at ambient temperature；Again After concussion is mixed, it is uniformly added into Tissue Culture Dish, is fully mixed dropwise immediately, puts in 37 DEG C of 5%CO2 cell culture incubators and train Support.

The screening of recombinant virus and plaque purification：During cytopathy to appear with Positive fluorescence signal, multigelation After cell culture fluid, 4 DEG C of 4500r/min centrifugations l0min, supernatant is drawn.Supernatant is trained with the DMEM of serum-free antibiotic-free Support base and carry out 10 times of doubling dilutions.Take 1mL dilution factors 10-2-10-7 poison disease vaccination grown in 6 porocyte culture plates it is good 1h is acted under the conditions of good PK-15 cell monolayers, 37 DEG C of 5%CO2.Washed 3 times with the DMEM culture mediums of serum-free antibiotic-free. By 2% low melting-point agarose heating and melting, it is placed in cooling down in 37 DEG C of water-baths, while 20%2 × DMEM culture mediums are also put In 37 DEG C of water-baths, after temperature is consistent, by 1:1 ratio is mixed.The mixed agarose gels of 2mL, 4 DEG C of placements are added per hole 10min, is cultivated after being transferred to Tissue Culture Plate in 37 DEG C of CO2 cell culture incubators after agarose gel solidification.Show in being inverted fluorescence Micro- Microscopic observation simultaneously marks the single plaque with green florescent signal, draws and green fluorescence letter is presented under maximum dilution multiple Number plaque, blown and beaten in DMEM nutrient solution of the 1mL serum-frees without antibiotic and carry out multigelation.Grasped more than repeating Make, the plaque liquid of acquisition is subjected to many wheel plaque purifications.The recombinant virus of acquisition is named as rPRV-GFP-gE-.Reference picture 4a, Fig. 4 b be the green florescent signal that observes under the CPE that is produced on PK-15 cells of recombinant virus and fluorescence microscope its As can be seen that recombinant virus is entered, PK-15 is intracellular in middle Fig. 4 a, and expression is active.

(3) PRV gE gene-deleted strains C1201/ Δs gE acquisition and purifying

Recombinant virus rPRV-GFP-gE- genome is extracted, Cre restructuring ferment treatments are carried out according to following system：2μL 10 × Cre Buffer, 10 μ g target dnas, 5 μ L Cre enzymes (1UI/ μ L), supplement ultra-pure water to 30 μ L.Mix, put 37 DEG C of water-baths 1h, then put 70 DEG C of processing 10min.

The viral DNA after Cre ferment treatments is extracted, concrete operation method is as follows：Add in reaction system after Cre ferment treatments Enter 200 μ L phenol：Chloroform：Isoamyl alcohol (25:24:1) extract 2 times, supernatant is taken after centrifugation；The absolute ethyl alcohol of 500 μ L precoolings is added, - 20 DEG C of precipitation 10min, 12000rpm/min centrifugation 15min are put, supernatant is abandoned；Add in the rearmounted incubator of 70% ethanol washing precipitation Dry.Gained is precipitated and dissolved in appropriate TE solution, and its concentration is determined with ultraviolet specrophotometer, puts -20 DEG C of preservations.

Sterilizing EP pipes in add 500 μ L Opti-MEM, the viral DNA and 6 μ L PLUS that 6 μ L Cre ferment treatments are crossed, mix 5min is stood after even；12 μ L LTX are added, 30min is stood after mixing.The vigorous Vero cells of growth selection.Mixed liquor is complete Portion is added in cell culture fluid, is put in 37 DEG C of 5%CO2 incubators and is cultivated.5%DMEM nutrient solutions are changed after 12h.CPE is observed, and In fluorescence microscopy Microscopic observation green fluorescence production, CPE is marked substantially but not it was observed that the region of obvious fluorescence signal. The cell of selection area is drawn, is put in 5%DMEM nutrient solutions.Obtained virus liquid is subjected to plaque purification, the gene of acquisition lacks Lose strain and be named as PRV C1201/ Δs gE.The PRV C1201/ Δs gE that reference picture 5a- Fig. 5 d are shown after purification is thin in Vero CPE and indirect immunofluorescene assay result on born of the same parents.

(4) PRV C1201/ Δs gE sequencing

Extract PRV C1201/ Δs gE genome and using it as template, using gE-L F and gE-R R as primer, enter performing PCR Amplification.After PCR primer purifying is reclaimed, connect into pJET1.2/blunt Cloning Vector and convert Trans10 competence Cell, the monoclonal bacterium that PCR is accredited as the positive carries out sequencing.Sequencing result is shown, compared with parent's strain, PRV C1201/ Δs gE gene delection region is included between 872-1101 bit bases, US7 and the US8 of the US7 genes of coding gI albumen Gap areas and coding gE albumen whole US8 genes.

2. genetic stability is tested：

Inoculating cell, the strain that clone purification is got is primary, as P0 generations.6 generations are compared by sequencing with P0 Gene variation.

As a result show, the clinical symptoms and pathology cut open inspection difference of 6 generation viruses are not notable.Clinical animal test results table Bright, C1201/ Δs gE involves continuous recurrence animal in a criminal case, and toxicity is not returned by force.

Similar with classical Natural Avirulent Strain Bartha-k61, pseudorabies virus gene-deleted strain C1201/ Δs gE is present A large amount of nucleotide deletions, absent region includes part gI and whole gE, specially encodes the 872-1101 of the US7 genes of gI albumen The whole US8 genes in Gap areas and coding gE albumen between bit base, US7 and US8.The gene-deleted strain is through pig interior generation 5 After secondary, amplification aim sequence does not find any sequence insertion.

Whether the vaccine virus for further 5 generations of checking is morphed, and the PCR primer expanded is carried out cutting glue time Receive, PCR primer directly send sequencing company to carry out positive and negative both direction sequencing.As a result show, P0 viruses and other each generations are square To sequence homology in 99.9-100%, the sequence homology of opposite direction is in 99.8-100%.P0 is for gene-deleted strain C1201/ Δs gE and the sequence comparative result of Bartha-K61 plants of relevant positions show that the similitude at 3 ' ends is 56.6%, and 5 ' The similitude at end only 54.5%, it is not same strain to illustrate gE and Bartha-K61 plants of C1201/ Δs.P0 generation viruses are forward and reverse As shown in Figure 12 and Figure 13, the genetic distance result of forward direction sequencing is as shown in figure 14 for the peak figure of sequencing, the sequence ratio of forward direction sequencing As shown in figure 15 to result, as shown in figure 16, the sequence alignment result of backward sequencing is as schemed for the genetic distance result of backward sequencing Shown in 17.

3. inactivate immuning effect test

3.1 packet：Experiment is randomly divided into 3 immune groups (every group 5) with weanling pig, sets 1 to attack malicious right in addition According to group (5) and 1 blank control group (5).

3.2 inactivated vaccines are prepared：Viral level before inactivation is adjusted to 107.5TCID50/mL, and immunologic adjuvant 5:1 Piglet is immunized for preparation.

3.3 it is immune：The inactivated vaccine prepared is subjected to immune every pig musculi colli according to packet and vaccinates 2ml.

3.5 attack poison：To 3 immune groups and attack malicious control group to carry out PRV BJ-11 plant strong malicious within 28 days after immune (104.0TCID50/mL) is attacked, and attacking malicious method is：Every piglet musculi colli injects 3.0ml, while collunarium 3.0ml.Blank Control group does not attack poison.

3.6 attack observation after poison：Piglet attacks Continuous Observation 21 days after poison, measures body temperature daily, the appetite of viewing test piglet, The state of mind, have difficulty in breathing, have loose bowels, runny nose, death and whether there is clinical symptoms performance.

3.8 criterion

3.8.1 malicious sequela criterion is attacked：There is following (3) item, or occur situation (1) simultaneously and (2) are judged to Morbidity.

(1) spiritual depressed, anorexia or there is expiratory dyspnea (containing cough, breathe, open one's mouth breathing, abdominal respiration), flow Tears, have loose bowels；

(2) body temperature >=40.5 DEG C, continue at least 3 days；

(3) obvious nervous symptoms (incoordination, paralysis, four limbs are scarified, opisthotonos etc.) or dead.

4th, result

Poison is attacked within 28th after 4.1 vaccines are immune to the immune efficacy result of weanling pig, Continuous Observation 21 days, 3 batches of vaccines are exempted from Epidemic disease group protective rate is more than 80%.And the non-Immunization control group piglet incidence of disease is 5/5 (100%), wherein there is 2 mutual aid occur Imbalance, paralysis, four limbs such as scarify at the nervous symptoms；Separately there is 1 pig death.Blank control group is without any adverse reaction.Result of the test See Fig. 6, thermometric is recorded as shown in Figure 7.

Shown in reference picture 8a- Fig. 8 f, Fig. 9 a- Fig. 9 d, wherein Fig. 8 a, Fig. 8 c, Fig. 8 e are respectively lungs, spleen, tonsillotome Attack malicious group, Fig. 8 b, Fig. 8 d, Fig. 8 f are immune group, Fig. 9 a, Fig. 9 c be respectively attack poison group brain IHC and attack poison group spleen IHC, Fig. 9 b, Fig. 9 d are respectively immune group brain IHC and immune group spleen IHC, and cut open inspection result is：Attack after malicious control group morbid pig dissection, lung has sternly The canescence Necrotic Nodule of weight, bleeding, oedema, spleen have necrosis region, and hemorrhage of tonsil, brain, spleen IHC results are positive.It is other Immune swine cut open inspection lung, spleen, tonsillotome are without obvious pathological change.

5th, conclusion

Result of the test shows, is not less than 80% to the protective rate of new popular strain after the vaccine immunity weanling pig.Wean Piglet immunological can resist the attack of new popular strong HB-1201 plants of the poison of pseudorabies using the C1201/ Δs gE inactivated vaccines prepared.

Presently preferred embodiments of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned Particular implementation, wherein the equipment and structure be not described in detail to the greatest extent are construed as giving reality with the common mode in this area Apply；Any those skilled in the art, without departing from the scope of the technical proposal of the invention, all using the disclosure above Methods and techniques content make many possible variations and modification to technical solution of the present invention, or be revised as equivalent variations etc. Embodiment is imitated, this has no effect on the substantive content of the present invention.Therefore, every content without departing from technical solution of the present invention, foundation The technical spirit of the present invention still falls within the present invention to any simple modifications, equivalents, and modifications made for any of the above embodiments In the range of technical scheme protection.

Sequence table

<110>Biotechnology Co., Ltd. Shanghai Chuanghong

<120>A kind of gE plants of PRV gene-deleted strain C1201/ Δs 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

gggtcggacc 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.