CN107828741A

CN107828741A - The dual-gene missing low virulent strain of pseudorabies virus and its application

Info

Publication number: CN107828741A
Application number: CN201711083192.7A
Authority: CN
Inventors: 王继春; 宋增财; 郭容利; 乔永峰; 王志胜; 许梦微; 刘娅梅; 郑亚婷; 范红杰; 侯继波
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2017-11-07
Filing date: 2017-11-07
Publication date: 2018-03-23
Anticipated expiration: 2037-11-07
Also published as: CN107828741B

Abstract

The present invention provides the dual-gene missing low virulent strain of pseudorabies virus and its application, belongs to the vaccines arts of animal medicine.80 plants of pseudorabies virus LA1206, deposit number are：CGMCC No.14329.The present invention also provides the dual-gene application for lacking low virulent strain in pseudorabies vaccines are prepared of pseudorabies virus, a kind of recombinant vector live vaccine.The recombinant vector obtains after external source protective antigen gene expression cassette is inserted in the low virulent strain genome.The live vaccine that 80 plants of PRV LA1206, available for the early immune of newborn piglet, blocks the infection of wild poison as early as possible to the safety of 1 age in days newborn piglet 100%.Can quickly produce efficient protection, the especially vaccine after inoculation wean PRV feminine gender piglets can not only prevent to fall ill, moreover it is possible to prevent toxin expelling.By being monitored to PRV gE and gB antibody, it is very beneficial for the purification to PRV variants.

Description

The dual-gene missing low virulent strain of pseudorabies virus and its application

Technical field

The invention belongs to the vaccines arts of animal medicine, and in particular to pseudorabies virus it is dual-gene missing low virulent strain and its Using.

Background technology

Pseudoabies is a kind of acute, strong biography caused by pseudorabies virus (Pseudorabies Virus, PRV) Catch an illness.The classical symptom that pseudoabies shows as very itching often, and heating and brain ridge occur for the domestic animals such as ox, sheep and wild animal The clinical symptoms such as marrow inflammation.And the symptom that pseudoabies occurs for pig is age-related, sow is often miscarried；Newborn piglet has nerve Symptom, the death rate are high；Growing and fattening pigs are mainly respiratory symptom, and Adult Pig infects growth retardation after Pseudorabies virus, do not sent out generally Life and death is died.Invention and large-scale promotion due to PRV gene delections strain vaccine (the Bartha K61 strains of such as gE gene delections) Use, and coordinate and differentiate immune swine and the foundation and application of the ELISA detection method of the PRV gE antibody of wild virus infection pig, pass through Positive boar is persistently eliminated, many countries realize the purification of the pseudorabies virus in domestic pig group in the world, in China, are permitted More pig farms have also been successfully established the swinery of PRV gE negative antibodies, and many pig farms realize PRV purification, porcine pseudorabies substantially Preferable control is obtained.

Since 2011, there is pig puppet on a large scale in many pig farms that traditional pseudorabies disease vaccine was immunized of northern China Rabies, epidemic situation is rapidly to the most of pig farm sprawling in the whole nation afterwards, and PRV gE antibody positive rates remain high, to China Pig industry causes to seriously endanger.The result of study of multiple team proves that this wheel epidemic situation is caused by the PRV street strains to make a variation, the change The pathogenic of different strain significantly increases, its major antigen gene have with traditional vaccine strain it is significantly different, for more effectively tackle PRV become The harm of different strain, protection can be improved using variant development, and can antidiastole immune swine is new with wild virus infection pig Vaccine is the key of current China's porcine pseudorabies prevention and control.But the security of existing variation strain live vaccine is high not enough.Example Such as, disclosed in the patent of invention of Application No. 201510388390.9 by knocking out the wild malicious AH02LA strains gE of pseudorabies virus The LA-A strains obtained after gene, the vaccine prepared using the strain is to the protecting effect of pseudorabies virus variant 100%, especially It is that the vaccine can not only prevent to fall ill, moreover it is possible to prevent toxin expelling, still, the strain is only to 4 week old piglets safety.

The content of the invention

It is an object of the invention to provide the dual-gene missing low virulent strain of pseudorabies virus, the strain can be good at adapting to ST Cell, titre is high, and virulence significantly reduces, and to 1 age in days piglet no pathogenicity, therefore is to have the vaccine candidate of higher-security weak Strain.

The dual-gene missing low virulent strain of pseudorabies virus, is pseudorabies virus LA1206-80 strains, and deposit number is： CGMCC No.14329。

It is a further object of the present invention to provide the dual-gene missing low virulent strain of pseudorabies virus to prepare pseudorabies vaccines In application, using immune pig can be provided 100% protection after the vaccine immunity, can not only prevent to fall ill, moreover it is possible to prevent Toxin expelling, because the strain virulence significantly reduces, to 1 age in days piglet no pathogenicity, therefore it can be used for the early immune of newborn piglet, The infection of wild poison is blocked as early as possible, and the live vaccine has higher security.

Another object of the present invention is to provide the vaccine using the dual-gene missing low virulent strain of pseudorabies virus as active component. Using the vaccine of pseudorabies virus LA1206-80 strains to 1 age in days newborn piglet safety, the negative susceptible piglets of inoculation wean PRV Can reach within 7 days afterwards can not only prevent to fall ill to the protecting effect of pseudorabies virus variant 100%, the especially vaccine, also Toxin expelling can be prevented, unexpected technique effect is all achieved in security and immune efficacy.By being monitored to antibody, Animal and the infection animal of the immune vaccine can be distinguished, is advantageous to the purification to PRV variants.

The present invention also provides a kind of recombinant vector live vaccine, and its active component is the dual-gene missing of the pseudorabies virus Insert what is obtained after external source protective antigen gene expression cassette in low virulent strain genome.Promoter can select pMCMV IE, SV40 and pHCMV IE etc., the insertion point of foreign gene includes between the UL22 and UL21 of genome, between UL46 and UL27, Between UL51 and UL50, between UL35 and UL36, between UL40 and UL41, between UL44 and UL26.5 between UL4 and UL3.5 Noncoding region, the foreign gene of insertion includes the E protein gene of CSFV, the Cap protein gene of pig circular ring virus, mad dog The G-protein gene of sick virus, the VP1 genes of foot and mouth disease virus, the VP60 genes of the HA genes of swine influenza virus and Rabbit pest virus Deng.

Live vaccine of the present invention, it is using pseudorabies virus LA1206-80 strains as active component, can be added in vaccine immune Adjuvant and vaccine excipients etc., those skilled in the art can easily be produced.

Applicant obtains one plant of pseudorabies virus gene delection low virulent strain by a large amount of creative work, That is PRV LA1206-80 strains.LA1206-80 strain gC and gD genes and completely the same (the gC genes GenBank of AH02LA strains: KR605320；GD genes GenBank:KR605321), with now disclosing variation strain's very high homology.Pseudoabies disease of the present invention Malicious LA1206-80 strains are first by PRV AH02LA strain gE genes (GenBank:KR605321) the 13rd~1298 bit base strikes Remove, be further continued for knocking out TK genes (SEQ ID No:1) the 184th~530 bit base, then weak rear clone is caused to screen through passage And obtain.Pseudorabies virus LA1206-80 strains can be well adapted for ST cells, and growth titre is up to 10^8.5TCID₅₀/mL.It is pseudo- The live vaccine of hydrophobin LA1206 strains is to the safety of 1 age in days newborn piglet 100%, available for the early immune of newborn piglet, The infection of wild poison is blocked as early as possible.Efficient protection can be quickly produced after inoculation wean PRV feminine gender piglets, pseudorabies virus is become Different strain 100% is protected, and especially the vaccine can not only prevent to fall ill, moreover it is possible to is prevented toxin expelling, is achieved unexpected technology effect Fruit.By being monitored to PRV gE and gB antibody, animal and the infection animal of the immune vaccine can be distinguished, is very beneficial for Purification to PRV variants.It is used as the protective antigens of the live vector expression susceptible cause of disease of pig, moreover it is possible to reach one pin of inoculation and prevent The effect of two or more epidemic diseases.

Brief description of the drawings

Fig. 1, transfer vector pPRV (AH02LA)-GFP (gE-) structure chart.

Fig. 2, transfer vector pPRV (AH02LA)-gE Δs structure chart.

Fig. 3, pseudorabies virus PRV AH02LA (Δ gE) strain structure schematic diagram.(A) PRV AH02LA strains are shown Genome, U_LRefer to unique long area, U_sRefer to unique short area, IR and TR refer to internal and end duplicate block respectively.(B) it show by upper Trip homology arm Δ gE-H1 and downstream homology arm Δ gE-H2 carries out the partial order that homologous recombination replaces GFP expression cassettes gE genes Row.(C) it show and knocks out GFP expression cassettes again by homologous recombination, obtains missing gE genes the 13rd~1298 altogether PRV AH02LA (Δ gE) strain of 1286bp nucleotides.

Fig. 4, transfer vector pPRV (AH02LA)-GFP (TK-) structure chart.

Fig. 5, transfer vector pPRV (AH02LA)-TK Δs structure chart.

Fig. 6, pseudorabies virus LA1206 strains structure schematic diagram.(A) it show the gene of PRV AH02LA (Δ gE) strain Group, U_LRefer to unique long area, U_sRefer to unique short area, IR and TR refer to internal and terminal repeat respectively.(B) it show same by upstream Source arm Δ TK-H1 and downstream homology arm Δ TK-H2 carries out the partial sequence that homologous recombination replaces GFP expression cassettes TK genes. (C) it show and knocks out GFP expression cassettes again by homologous recombination, obtains missing gE the 13rd~1298 common 1286bp cores of gene The LA1206 strains of the 184th to 530 common 347bp nucleotides of thuja acid and TK genes.

The growth characteristics of cultured of Fig. 7, LA1206-80 strain and parental virus compares.A：Infect on ST cell postoperative infection cells Viral titre in clear liquid；B：Cell-associated viral virus titer after infection ST cells.

Embodiment

The present invention is further illustrated below by way of specific embodiment, but scope and spirit of the present invention are not limited to listed reality Apply example.

Embodiment one, the structure of pseudorabies virus gE gene-deleted strains and identification

1st, the acquisition of transfer vector

It is artificial synthesized homologous by gE upstream region of gene to knock out the part gE genes of pseudorabies virus PRV AH02LA strains Arm Δ gE-H1, egfp expression box and gE downstream of gene homology arm Δs gE-H2 compositions genetic fragment, then should Fragment is cloned into pUC57 carriers (Nanjing Genscript Biotechnology Co., Ltd.), obtain GFP transfer vectors pPRV (AH02LA)- GFP (gE-), its structure are as shown in Figure 1.GE upstream region of gene homology arm Δs gE-H1 sequence such as SEQ ID No：Shown in 2, gE bases Because of downstream homology arm Δ gE-H2 sequence such as SEQ ID No：Shown in 3, the same Application No. of sequence of GFP expression cassettes 201510388390.9 patent of invention.

Then it is artificial synthesized to be made up of gE upstream region of gene homology arm Δ gE-H1 and gE downstream of gene homology arm Δs gE-H2 Genetic fragment, the fragment is cloned into pUC57 carriers (Nanjing Genscript Biotechnology Co., Ltd.), obtains transfer vector pPRV (AH02LA)-gE Δs, its structure are as shown in Figure 2.GE upstream region of gene homology arm Δs gE-H1 sequence such as SEQ ID No：Shown in 2, GE downstream of gene homology arm Δs gE-H2 sequence such as SEQ ID No：Shown in 3.

2nd, the acquisition of GFP recombinant pseudorabies virus AH02LA strains

Primary chicken embryo fibroblasts (Chicken embryo are prepared using 9~10 age in days SPF chicken embryos Fibroblasts, CEF), after cell grows up to individual layer, transfected.When being transfected according to conventional calcium phosphate infection protocol, by 0.5 ~1 μ g GFP transfer vectors pPRV (AH02LA)-GFP (gE-) the DNA and DNA of 0.2~1 μ g pseudorabies virus AH02LA strains The primary CEF of cotransfection, homologous recombination (Fig. 3) is carried out, nutrient solution is absorbed after 24 hours, addition contains 10% hyclone and 0.5% Methylcellulose DMEM culture mediums cover, and continue 24~48h of culture, and picking sends green in the case where wavelength is 488nm ultraviolet excitations The recombinant virus plaque of color fluorescence, is inoculated into fresh CEF, so repeats more than 3 circulations, obtains the recombinant virus of purifying, It is named as PRV AH02LA (Δ gE)-GFP strains.Using the DNA of PRV AH02LA (Δ gE)-GFP strains as template, using pair of primers (sense primer PRV gE site ckeck F sequences are:5’-TCTGGAGGGGCCCTCGCCGA-3’；Anti-sense primer PRV gE Site ckeck R sequences are:5 '-AGAGAGAGGACGGAGGCGTGTCATC-3 ') PCR amplification by gE upstream region of gene homology arms (Δ gE-H1), the glimmering protein expression box (GFP) of green and gE downstream of gene homology arm composition genetic fragment (Δ gE-H2), will expand Increase production thing and carry out electrophoresis and sequencing, to verify whether sequence is correctly inserted into.PCR reaction systems (table 1) and response procedures are such as Under.

The μ l PCR reaction systems of table 1 25

LA Taq archaeal dna polymerases, 2 × GC Buffer II in PCR reaction systems are purchased from Takara companies.

Reaction system is mixed, enters performing PCR amplification, response procedures are as follows：94 DEG C of pre-degeneration 4min；94 DEG C denaturation 40s, 57 DEG C annealing 40s, 72 DEG C extension 4min30s, 30 circulation；72 DEG C of extension 10min.PCR primer is subjected to 1% Ago-Gel electricity Swimming, detects whether 4585bp or so purpose band and is cut, and is reclaimed, will reclaimed using DNA gel QIAquick Gel Extraction Kit Product send Huada gene company to be sequenced.

After qualification result shows that recombinant virus PRV AH02LA (Δ gE)-GFP strains enter performing PCR amplification using above-mentioned primer, 4585bp or so purpose product is obtained, sequencing results are correct, and the recombinant virus is by pseudorabies virus AH02LA The 13rd to 1298 common 1286bp nucleotides of strain gE genes, which are replaced, inserts what GFP expression cassettes (Fig. 3) obtained afterwards, complete with expection It is complete consistent.

3rd, the acquisition and identification of pseudorabies virus gE gene-deleted strains

Take the DNA and 700~2000ng of 1~3 μ g recombinant viruses PRV AH02LA (Δ gE)-GFP strains pPRV (AH02LA) DNA of-gE Δs, according to conventional calcium phosphate infection protocol Transfected primary CEF, second of homologous recombination is carried out, to knock out GFP expression cassettes (Fig. 3).Absorb nutrient solution within 24 hours after cotransfection, add containing 10% hyclone and 0.5% methylcellulose DMEM culture mediums cover, and continue 24~48h of culture, and in the case where wavelength is 488nm ultraviolet excitations, picking does not send green fluorescence Virus plaques are inoculated with fresh CEF, so repeatedly 3~5 circulations, obtain virus after purification, are named as PRV AH02LA (Δs GE) strain, and as kind of a poison.The DNA of PRV AH02LA (Δ gE) strain is extracted as template, using primer PRV gE site Ckeck F and PRV gE site ckeck R enter performing PCR amplification, detect whether 2937bp or so purpose band and by its Cut, reclaimed using DNA gel QIAquick Gel Extraction Kit, and carry out sequencing.

As a result show, the special of 2900~3000bp is obtained using PRV AH02LA (Δ gE) strain DNA as template PCR amplifications Property fragment, sequencing result is with expected consistent, i.e. the 13rd to 1298 common 1286bp nucleotides of gE genes are by successful knockout.

Embodiment two, the structure of the dual-gene gene-deleted strain of pseudorabies virus gE, TK and identification

1st, the acquisition of transfer vector

To continue to knock out the part of TK gene of pseudorabies virus PRV AH02LA (Δ gE) strain, send Nanjing Jin Sirui biological Science and Technology Ltd. is artificial synthesized by TK upstream region of gene homology arm Δs TK-H1, egfp expression box (GFP) and TK genes The genetic fragment of downstream homology arm Δ TK-H2 compositions, is then cloned into pUC57 carriers by the fragment, obtains GFP transfer vectors PPRV (AH02LA)-GFP (TK-), its structure are as shown in Figure 4.TK upstream region of gene homology arm Δs TK-H1 sequence such as SEQ ID No：Shown in 4, TK downstream of gene homology arm Δs TK-H2 sequence such as SEQ ID No：Shown in 5, the sequence of GFP expression cassettes is the same as application Number it is 201510388390.9 patents of invention.

Send Nanjing Genscript Biotechnology Co., Ltd. artificial synthesized by TK upstream region of gene homology arm Δ TK-H1 and TK genes The genetic fragment of downstream homology arm Δ TK-H2 compositions, is then cloned into pUC57 carriers by the fragment, obtains transfer vector pPRV (AH02LA)-TK Δs, its structure are as shown in Figure 5.

2nd, the acquisition of GFP recombinant pseudorabies virus PRV AH02LA (Δ gE) strain

Primary chicken embryo fibroblasts (Chicken embryo are prepared using 9~10 age in days SPF chicken embryos Fibroblasts, CEF), after cell grows up to individual layer, transfected.When being transfected according to conventional calcium phosphate infection protocol, by 0.5 ~1 μ g GFP transfer vectors pPRV (LA-A)-GFP (TK-) DNA and 0.2~1 μ g pseudorabies virus PRV AH02LA (Δ gE) The primary CEF of DNA cotransfections of strain, homologous recombination (Fig. 6) is carried out, nutrient solution is absorbed after 24 hours, addition contains 10% hyclone Covered with the DMEM culture mediums of 0.5% methylcellulose, continue 24~48h of culture, picking is 488nm ultraviolet excitations in wavelength Under send the recombinant virus plaque of green fluorescence, be inoculated into fresh CEF, so repeat more than 3 circulations, obtain the weight of purifying Group virus, is named as PRV AH02LA (gE-)-GFP (TK-) strain.Using the DNA of PRV AH02LA (gE-)-GFP (TK-) strain as mould Plate, using pair of primers, (sense primer PRV Δ TK ckeck F sequences are:5’-CGCACCCCGAGGTTGACTTCAA-3’；Under Swimming primer PRV Δ TK ckeck R sequences is:5 '-TTGTACGCGCCGAAGAGGGTGT-3 ') PCR amplification by TK upstream region of gene The gene piece of homology arm (Δ TK-H1), the glimmering protein expression box (GFP) of green and TK downstream of gene homology arms (Δ TK-H2) composition Section, amplified production is subjected to electrophoresis and sequencing, to verify whether sequence is correctly inserted into.PCR reaction systems (table 2) and reaction Program is as follows.

The μ l PCR reaction systems of table 2 25

Reaction system is mixed, enters performing PCR amplification, response procedures are as follows：94 DEG C of pre-degeneration 4min；94 DEG C denaturation 40s, 58 DEG C annealing 40s, 72 DEG C extension 2min30s, 30 circulation；72 DEG C of extension 10min.PCR primer is subjected to 1% Ago-Gel electricity Swimming, detects whether 2200bp or so purpose band and is cut, and is reclaimed, will reclaimed using DNA gel QIAquick Gel Extraction Kit Product send Huada gene company to be sequenced.

Qualification result shows that recombinant virus PRV AH02LA (gE-)-GFP (TK-) strains enter performing PCR amplification using above-mentioned primer Afterwards, 2200bp or so purpose product has been obtained, sequencing results are correct, and the recombinant virus is by pseudorabies virus PRV The common 347bp nucleotides replacement of TK genes the 184th to 530 of AH02LA (Δ gE) strain obtains after inserting GFP expression cassettes (Fig. 6) , it is completely the same with expection.

3rd, the acquisition and identification of the dual-gene gene-deleted strains of pseudorabies virus gE, TK

Take the DNA and 700~2000ng of 1~3 μ g recombinant virus PRV AH02LA (gE-)-GFP (TK-) strain pPRV (AH02LA) DNA of-TK Δs, according to conventional calcium phosphate infection protocol Transfected primary CEF, second of homologous recombination is carried out, to knock out GFP expression cassettes (Fig. 6).Absorb nutrient solution within 24 hours after cotransfection, add containing 10% hyclone and 0.5% methylcellulose DMEM culture mediums cover, and continue 24~48h of culture, and in the case where wavelength is 488nm ultraviolet excitations, picking does not send green fluorescence Virus plaques are inoculated with fresh CEF, so repeatedly 3~5 circulations, obtain virus after purification, are named as PRV LA1206 strains, and As kind of a poison.The DNA of PRV LA1206 strains is extracted as template, using primer PRV Δ TK ckeck F and PRV Δ TK ckeck R enters performing PCR amplification, detects whether 347bp purpose band and is cut, and is reclaimed using DNA gel QIAquick Gel Extraction Kit, and Carry out sequencing.

As a result show, 300~400bp specific fragment obtained using PRV LA1206 strains DNA as template PCR amplifications, Sequencing result is with expected consistent, i.e. the 184th to 530 common 347bp nucleotides quilts of TK genes of PRV AH02LA (Δ gE) strain Successful knockout.

4th, the passage of pseudorabies virus LA1206 strains kind poison

Further to improve the security of pseudorabies virus LA1206 strains, pseudorabies virus LA1206 strain kind poison is existed Continuous passage is carried out on CEF, every 5 generation preserves batch of seeds, passed to for the 120th generation altogether, wherein from the 75th generation to the 80th generation system through even Continue 5 generation picking single virus Plaque Clones and obtain F80 generations.The malicious 1st generation of pseudorabies virus LA1206 strain kinds is designated as pseudoabies The viral malicious F1 generation of LA1206 strains kind, other the like.The seed that each generation preserves is put -70 DEG C and stored for future use.

5th, the measure of the gC and gD gene orders of pseudorabies virus LA1206 strains

Type B error hydrophobin LA1206 strain kinds malicious F2 generations and the DNA in F80 generations, itself gC and gD gene piece is expanded by PCR Section, and carry out sequencing.PRV ZJ01 strains first in GenBank

(GenBank is logged in：KM061380.1) whole genome sequence, using Vector NTI softwares, 2 couples of PCR are designed altogether Primer (such as table 3), the amplified fragments of these primers cover PRV main protection antigen gC and gD gene order.It is all to set The primer of meter is synthesized by Nanjing Genscript Biotechnology Co., Ltd..

Table 3 expands the PCR primer of PRV gC and gD genes

Using the DNA in pseudorabies virus LA1206 strain kinds malicious F2 generations and F80 generations as template, gC and gD gene sequences are expanded respectively Row.Primer in PCR reaction systems as shown in table 1 is replaced with to the primer in table 3, other are constant.PCR reaction conditions are：94 ℃1min；94 DEG C of 30s, annealing 30s (determining annealing temperature according to primer Tm), 72 DEG C of extensions are (true according to amplified fragments size Determine extension of time), 30 circulations；72 DEG C of extension 5min.After the completion of PCR, with 1% agarose gel electrophoresis identify, cut containing PCR purpose band gel piece of the same size with expection carries out DNA recovery, send 2 glycoprotein gene fragments of recovery to China Big genome company is sequenced.

Sequencing result shows that pseudorabies virus LA1206 strain kinds malicious F2 generations are identical with gC with the gD gene orders in F80 generations, And it is identical with AH02LA strains, there is the characteristic feature of PRV variants, and any variation does not occur in passing on.GC and gD is pseudo- mad The main protection antigen of dog disease virus, its gene show pseudorabies virus LA1206 strains kind poison not because passage changes Immunogenicity completely kept.

In pseudorabies virus LA1206 strain kinds malicious F80 generations, are named as pseudorabies virus LA1206-80 strains, i.e. PRV LA1206-80 strains.

Embodiment three, the identification of the growth characteristics of PRV LA1206-80 strains

By velogen strain PRV AH02LA strains, PRV LA-A strains and PRV LA1206-80 strains respectively using MOI as 0.004 inoculation Cover with the ST cells of individual layer, put 5%CO₂In constant incubator after 37 DEG C of incubation 1h, supernatant is sucked, is washed 3 times using PBS Afterwards, cell maintenance medium (the DMEM culture mediums (Gibco) for containing 3% hyclone (FBS)) is changed into be cultivated, and respectively at inoculation 0h, 6h, 12h, 24h, 36h, 48h and 72h determine in infection cell supernatant that cell combines in free virus and infection cell afterwards The content of venereal disease poison.

The free virus content of infection cell supernatant is to be operated in accordance with the following methods：At the appointed time put and receive respectively Collect infection cell supernatant 1ml, 376 × g centrifugation 5min and take 100 μ l supernatants, add in 900 μ l DMEM culture mediums and mix, then 100 μ l are therefrom drawn into 900 μ l DMEM culture mediums, repeat above step, i.e., 10~10 are made to infection cell supernatant⁹Times Than dilution.Viral dilution liquid is inoculated in 96 porocyte plates of the individual layer ST cells being paved with advance, each dilution factor makees 8 weights It is multiple, 37 DEG C of incubation 2h, virus liquid is discarded, (DMEM containing 3% hyclone (FBS) is trained 100 μ l of addition cell maintenance medium per hole Support base (Gibco)).Start to observe cytopathy situation after 12h, Continuous Observation 3 days, virus is calculated according to Reed-Muench methods TCID₅₀, count the TCID of all time point virus liquids₅₀, draw growth curve.

The cell-associated viral level detection of infection cell is operated in accordance with the following methods：Suck cell supernatant, With PBS infection cell 2 times, be resuspended after scraping cells with 2ml DMEM, -70 DEG C with 500 × g after 37 DEG C of multigelations 3 times 5min is centrifuged, takes 100 μ l supernatants to make 10~10⁹Doubling dilution.ST is inoculated with by above-mentioned free virus content detection same procedure Cell simultaneously determines viral TCID₅₀, count the TCID of all time point virus liquids₅₀, draw growth curve.

The dual-gene gene-deleted strain low virulent strain PRV LA1206- of TK, gE are can be seen that from the growth in vitro curve (Fig. 7) of virus 80 plants have similar growth kinetics to gE gene delection virus PRV LA-A strains and parental virus PRV AH02LA strains, are connecing 24h~48h reaches peak value after kind, although PRV LA1206-80 strains are declined slightly compared with LA-A strains and AH02LA strains, titre is still Reach 10^8.0TCID₅₀/ more than mL, the requirement of live vaccine production can be met.

Example IV, the security of PRV LA1206-80 strains

1st, test material

It is 1.1 viral

PRV LA1206-80 strains are inoculated with ST cells, cultivated using the DMEM containing 3% (concentration expressed in percentage by volume) hyclone Base culture 40 hours, viral cultures are harvested, centrifuging and taking supernatant is as PRV LA1206-80 strain virus liquid after freeze thawing.

The pseudorabies virus malicious F2 of LA1206 strain kinds for virus liquid (preparation method is with PRV LA1206-80 strain virus liquid) and PRV LA1206-80 strain virus liquid, viral level is respectively 10^8.0TCID₅₀/ mL and 10^8.1TCID₅₀/mL；PRV AH02LA strains Virus liquid, viral level 10^9.0TCID₅₀/mL；PRV LA-A strain virus liquid, viral level 10^8.3TCID₅₀/mL.It is viral equal Put -70 DEG C of preservations.

1.2 diagnostic kit

PRV gE antibody diagnosing reagent kits, CIVTESTSUIS ADVgB HIPRA Products.PRV gB antibody diagnosis tries Agent box, CIVTESTSUIS ADVgB HIPRA Products.

2nd, experimental animal

Experiment piglet is brood 1 age in days sodium selenite, and its PRV antibody is feminine gender.Diet, the essence of pig are examined before inoculation Refreshing situation and clinical setting etc., all unsound pigs exclude experiment.It is identified by ear tag number and cage number.With lactation Sow is raised in isolation environment jointly.Special messenger is responsible for raising, carries out 2 sanitation and hygiene daily.

Test mice is 17~22 grams of Kunming system cleaning grade mouse, purchased from Shanghai Jie Sijie experimental animals Co., Ltd.Raise Support in clean environment, special messenger raising, daily sanitation and hygiene.

3rd, it is grouped

Brood 1 age in days tests piglet, is randomly divided into 3 groups (A-1, A-2, A-3), every group 4, carries out PRV LA1206 strain kinds The safety testing in malicious F2 generations, PRV LA1206-80 strains and the inoculation of PRV LA-A strains collunarium.Another age in days of nest 1 tests piglet, with Machine is divided into 3 groups (B-1, B-2, B-3), every group 4, carries out PRV LA1206 strain kinds malicious F2 generations, PRV LA1206-80 strains and PRV The safety testing of LA-A strain intramuscular inoculations.

Test mice is randomly divided into 20 groups, every group 4, carries out PRV AH02LA strains, PRV LA-A strains, PRV LA1206 strains Kind malicious F2 generations, the safety testing of PRV LA1206-80 strains, specific group technology are shown in Table 4.

Table 4, test mice packet

4th, test method

4.1 brood 1 age in days piglet inoculation methods and clinical observation

DMEM culture mediums are applied into PRV LA1206 strain kinds malicious F2 generations, PRV LA1206-80 strains and PRV LA-A strains respectively It is diluted to 10^7.0TCID₅₀/ mL, it is each in a manner of collunarium inoculation and intramuscular inoculation respectively to be inoculated with 1 age in days piglet one group (4 Head), inoculum concentration is 1mL/ heads, is specifically shown in Table 5.From inoculation day to after being inoculated with 14 days, the body temperature of daily measurement experiment piglet, observation Diet and mental status and clinical symptoms.

4.2 mouse inoculation methods and observation

By PRV AH02LA strains and PRV LA1206 strain kinds malicious F2 generations, PRV LA1206-80 strains and PRV LA-A strains point Not Ying Yong DMEM culture mediums be diluted to 10^6.0TCID₅₀/ 0.2mL, then suitable concn is diluted to DMEM culture mediums respectively, with subcutaneous Injection system is inoculated with, each virus inoculation amount such as table 4, and the inoculation volume of virus liquid is 0.2mL/.From inoculation day to after being inoculated with 14 My god, viewing test dead mouse situation.The median lethal dose to mouse is calculated by Reed and Muench methods.

5th, result of the test

5.1 1 age in days piglets results

Each strain the results are shown in Table 5 to the safety testing of piglet.The PRV LA-A strains of missing gE genes resist to 1 age in days PRV Body feminine gender piglet still has stronger pathogenicity, after TK and gE Gene Doubles missing is carried out its (i.e. PRV LA1206 strains kind malicious F2 generations) Pathogenicity is remarkably decreased, but either collunarium inoculation or intramuscular injection inoculation all causes the body temperature of test pig to raise, and 4 test pigs In there is 1 hair to die of illness to die (table 4), show still there is virulence, and after continuous passage to F80 generations, either collunarium or flesh Note inoculation does not all cause experiment temperature of pig body rise, and no morbidity is dead, shows continuous passage further by the dual-gene missings of TK and gE Strain causes weak, it was demonstrated that PRV LA1206-80 strains have good security.

The safety testing packet of the age in days piglet of table 51 and result

5.2 mouse test results

LD of the PRV AH02LA strains to mouse₅₀For 10^2.57TCID₅₀, LD of the PRV LA-A strains to mouse₅₀For 10^4.96TCID₅₀, PRV LA1206 strain kinds malicious F2 generations and PRV LA1206-80 strains are with 10^6.0TCID₅₀Mice Inoculated does not cause extremely Die.

The preservation information of PRV LA1206-80 strains is as follows：

Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center.

Unit address：Institute of microbiology of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences.

Join the biomaterial (strain) of Ju：PRV LA1206-80.

Classification And Nomenclature：Porcine herpesvirus 1.

Preserving number：CGMCC No.14329.

Preservation date：On July 24th, 2017.

Embodiment five, the immune efficacy of PRV LA1206-80 strains

1st, test material

1.1 viruses and pseudorabies living vaccines

PRV LA1206-80 strain virus liquid, viral level 10^8.1TCID₅₀/mL。

Pseudorabies living vaccines (Bartha K61 strains), purchased from Nanjing Tianbang Bio-industry Co., Ltd., batch 150826.

PRV AH02LA strains, for challenge test, to the LD of 45 age in days PRV feminine gender pigs₅₀For 10^6.5TCID₅₀。

1.2 diagnostic kit

PRV gE antibody diagnosing reagent kits and PRV gB antibody diagnosing reagent kits, from the prosperous biotechnology of Beijing Ai Deshi members Co., Ltd (IDEXX) company.

2nd, experimental animal

28~35 age in days sodium selenites 24, its PRV antigen and PRV gE are feminine gender, pig breeding and breathing with gB antibody Distress syndrome virus (PRRSV), CSFV (SFV), porcine circovirus 2 type (PCV2) and O-shaped foot and mouth disease virus (FMDV (O)) antigen and antibody are feminine gender.Diet, mental status and clinical setting of pig etc., all unsound pigs are examined before inoculation Only exclude experiment.It is identified by ear tag number and cage number with circle number.The sub-cage rearing in isolation environment.Feed is full price Mixed feed (commercially available prod), drink water and respectively fed once for running water, the daily morning and afternoon.Special messenger is responsible for raising, enters daily 2 sanitation and hygiene of row.

3rd, it is grouped

Experimental animal once introduces, and is randomly divided into A~F groups, every group 4, is specifically shown in Table 6.

4th, test method and the index of research

(1) test method

It is immune：PRV LA1206-80 strain virus liquid and pseudorabies living vaccines (Bartha K61 strains) application is sterile DMEM nutrient solutions are diluted to suitable concentration, intramuscular injection, 2mL/ heads, A groups, B groups and component C and are not inoculated with 1000TCID₅₀、 10000TCID₅₀And 100000TCID₅₀PRV LA1206-80 strain virus liquid, D groups inoculation 100000TCID₅₀Pseudoabies Live vaccine (Bartha K61 strains), every intramuscular injection 2mL DMEM liquid (table 6) of E, F group.

Table 6, the packet of animal immune challenge test

Group	Immunizing dose	It is inoculated with strain
			A groups	1000TCID₅₀	PRV LA1206-80 strains
B groups	10000TCID₅₀	PRV LA1206-80 strains
			C groups	100000TCID₅₀	PRV LA1206-80 strains
D groups	100000TCID₅₀	Pseudorabies living vaccines (Bartha K61 strains)
			E groups	Blank control	/
F groups	Attack malicious control	/

Attack poison：All pigs in addition to blank control group attack poison in 1 week after inoculation, using PRV AH02LA strains, with 2LD₅₀/ Head collunarium attacks poison, every 2mL, is observed 14 days after attacking poison.

Serum gB and gE antibody test：Before immune, attack before poison and (attacked after poison 14 days) three time points at the end of attacking poison All pig blood serum samples are taken, PRV gE and gB antibody is detected using kit.

Clinicing symptom observation：All experiment pigs observe spirit, diet and various respiratory systems, nervous system daily With systemic clinical manifestation.

Measurement of bldy temperature：Since before inoculation, to off-test (attack poison after 14 days), all pigs measure body temperature once daily (rectal temperature).

Toxin expelling situation detects：Before attacking poison, attack after poison to off-test (attack poison after 14 days), gather pig nose swab sample daily Product, sample put -70 DEG C of preservations, are inoculated with BHK-21 cell observations lesion after doubling dilution to determine whether discharge virus during detection.

(2) index of research

The incidence of disease, the death rate, body temperature rising condition, toxin expelling situation, serum antibody sun turn situation.

5th, result of the test

PRV gB and the gE antibody of all test pigs is feminine gender before inoculation, and blank control pig (E groups) is to during off-test PRV gB and gE antibody be feminine gender, A groups (before attacking poison) at 1 week after inoculation, B groups, all test pigs of C groups and D groups PRV gB antibody is the positive, and it is the positive to attack all immune group pigs after poison and attack the PRV gE antibody of malicious control group survival pig (table 7).

Table 7, Immunization test pig serum antibody sun turn situation statistical form

After attacking poison, blank control group pig is dead without morbidity, no body temperature rise, does not detect toxin expelling；Attack malicious 4 pigs of control group All occur subtracting food since after attacking poison the 2nd day, spirit is depressed, sneezing, and symptom, the body temperature such as rhinorrhea and expiratory dyspnea all rise Height detects toxin expelling, the 4th~6 day dead successively 3 after poison is attacked to more than 40.5 DEG C from nose swab sample；Show to test (table 8) is set up in control.A groups, B groups, all test pigs of C groups without clinical symptoms, no body temperature rise, do not detect toxin expelling, show PRV LA1206-80 strains can produce protection completely to PRV variants；D groups test pig has 1 pig clinical symptoms occur without death, 4 There is body temperature rise in pig, detects toxin expelling from nose swab sample, shows that PRV Bartha K61 strains only produce part guarantor Protect (table 8).

Table 8, Immunization test pig clinical setting statistical form

In summary, the equal complete guarantor that can be produced to the AH02LA strains of PRV street strains of pig is immunized in PRV LA1206-80 strains Shield, as little as most 1000TCID₅₀Inoculum concentration it is immune after can also resist morbidity and toxin expelling, and 100000TCID₅₀PRV Bartha K61 strains can only generating unit code insurance shield, it is impossible to prevent body temperature react and toxin expelling, LA1206-80 strains are as vaccine strain to variant Protecting effect is significantly better than Bartha K61 strains.Live vaccination pig detection PRV gB prepared by PRV LA1206-80 strains resist Body, PRV gE antibody not being detected, being a kind of DIVA vaccines that can differentiate vaccine immunity animal and wild virus infection animal, it is promoted should With will be very beneficial for carrying out the purification of PRV variants in pig farm, have a extensive future.Due to LA1206-80 strains, virulence significantly drops It is low, to 1 age in days piglet no pathogenicity, therefore it is the vaccine candidate low virulent strain with higher-security, available for newborn piglet Early immune, the infection of wild poison is blocked as early as possible.

SEQUENCE LISTING

<110>Jiangsu Province Agriculture Science Institute

<120>The dual-gene missing low virulent strain of pseudorabies virus and its application

<130> 20171103

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 963

<212> DNA

<213>PRV AH02LA strains

<400> 1

atgcgcatcc tccggatcta cctcgacggc gcctacggca ccggcaagag caccacggcc 60

cgggtgatgg cgctcggcgg ggcgctgtac gtgcccgagc cgatggcgta ctggcgcact 120

ctgttcgaca cggacacggt ggccggtatt tacgatgcgc agacccggaa gcagaacggc 180

agcctgagcg aggaggacgc ggccctcgtc acggcgcagc accaggccgc cttcgcgacg 240

ccgtacctgc tgctgcacac gcgcctggtc ccgctcttcg ggcccgcggt cgagggcccg 300

cccgagatga cggtcgtctt tgaccgccac ccggtggccg cgacggtgtg cttcccgctg 360

gcgcgcttca tcgtcgggga catcagcgcg gcggccttcg tgggcctggc ggccacgctg 420

cccggggagc cccccggcgg caacctggtg gtggcctcgc tggacccgga cgagcacctg 480

cggcgcctgc gcgcccgcgc gcgcgccggg gagcacgtgg acgcgcgcct gctcacggcc 540

ctgcgcaacg tctacgccat gctggtcaac acgtcgcgct acctgagctc ggggcgccgc 600

tggcgcgacg actgggggcg cgcgccgcgc ttcgaccaga ccgtgcgcga ctgcctcgcg 660

ctcaacgagc tctgccgccc gcgcgacgac cccgagctcc aggacaccct cttcggcgcg 720

tacaaggcgc ccgagctctg cgaccggcgc gggcggccgc tcgaggtgca cgcgtgggcg 780

atggacgcgc tcgtggccaa gctgctgccg ctgcgcgtct ccaccgtcga cctggggccc 840

tcgccgcgcg tctgcgccgc ggccgtggcg gcgcaggcgc gcggcatgga ggtgacggag 900

tccgcgtacg gcgaccacat ccggcagtgc gtgtgcgcct tcacgtcgga gatgggggtg 960

tga 963

<210> 2

<211> 1023

<212> DNA

<213> artificial

<220>

<223>GE upstream region of gene homology arm Δs gE-H1

<400> 2

tgtttctgga ggggccctcg ccgagcaact acagcgggcg cgtggagctg ctgcgcctcg 60

accccaagcg cgcctgctac acgcgcgagt acgccgccga gtacgacctc tgcccccgcg 120

tgcaccacga agccttccgc ggctgcctgc gcaagcgcga gccgctcgcc cggcgcgcgt 180

ccgccgcggt ggaggcgcgc cggctgctgt tcgtctcgcg cccggcctcg ggggacgcgg 240

ggtcgtacgt gctgcgggtc cgcgtgaacg ggaccacgga cctctttgtg ctgacggccc 300

tggtgccgcc gagggggcgc cccgtcccca cgtcgccgcc cgcggacgag tgccggcccg 360

tcgtcggatc gtggcacgac agcctgcgcg tcgtggaccc cgccgaggac gccgtgttca 420

ccacccagcc cccgcccgag cccgagccgc cgacgacccc cgcgcccccc cgggggaccg 480

gcgccacccc cgagccccga tcggacgagg aggaggaggt gacgcggaga cgacgacgcc 540

gactgacccc cgcgcccggg accctggacg cgaacggcac gatggtgctg aacgccagcg 600

tcgtgtcgcg cgtcctgctc gccgccgcca acgccacggc gggcgcccgg agccccggga 660

agatagccat ggtgctgggg cccacgatcg tcgtcctcct gatcttcctg ggcgggatcg 720

cctgcgtggc ccggcgctgc gcgcggaatc gcatctaccg gccgcgaccc gggcgcggat 780

cggcggtcca tgcggcgccc ccgcggcgcc cgcccccaac cccggtcgcc ggggcgcccg 840

tcccccagcc caagatgacg ttggccgagc tgcgccagaa gctcgccacc atcgcagaag 900

aacaataaaa aggtggtgtt tgcataattt tgtgggtggc gttttatctc cgtccgcgcc 960

gttttaaacc tgggcacccc cgcgagtctc gcacacaccg gggttgagac catgcggccc 1020

ttt 1023

<210> 3

<211> 1113

<212> DNA

<213> artificial

<220>

<223>GE downstream of gene homology arm Δs gE-H2

<400> 3

gctggcgctg ggctccttcg tgatgacgtg cgtcgtcggg ggggccatct ggctctgcgt 60

gctgtgctcc cggcgccggg cggcctcgcg gccgttccgg gtgccgacgc gggcgcggac 120

gcacatgctc tctccggtgt acaccagcct gcccacgcac gaggactact acgacggcga 180

cgacgacgac gacgaggagg cgggcgtcat ccgccggcgg cccgcctccc ccagcggaga 240

cagcggctac gaggggccgt acgcgagcct ggaccccgag gacgagttca gcagcgacga 300

ggacgacggg ctgtacgtgc gccccgagga ggcgccccgc tccggcttcg acgtctggtt 360

ccgcgatccg gagaaaccgg aagtgacgaa tggacccaac tatggcgtga ccgccaaccg 420

cctgttgcat gtcccgcccc gctaaaatac cgggagaacc ggtccgcccg cattccgaca 480

tgcccggcgc cgcctccgtc gacatggaca cgtttgaccc cagcgccccc gtcccgacga 540

gcgtctcgaa cccggccgcc gacgtcctgc tggcccccaa gggaccccgc tccccgctgc 600

gcccccagga cgactcggac tgctactaca gcgagagcga caacgagacg cccagcgagt 660

tcctgcgccg cgtgggacgc cggcaggcgg cgcgtcggag acgccgccgc tgcctgatgg 720

gcgtcgcgat cagcgccgcc gcgctggtca tctgctcgct gtccgcgcta ctcgggggca 780

tcgtcgccag gcacgtgtag cgagcgagcg agcgaacggg agcgggggcc cgcccccatc 840

cgccgcgccc aggagagggg ggagagagcg gggggttggg cgcgccacgt ggtgtgggca 900

cggactcgga cttgtcacaa taaatgggcc ccggcgtgtc cgggcgcaca cagcagcctt 960

cctctcctcc gcgtctctgt tccgcccgtc tctcgccgga ctcttcttct ccaccgcctc 1020

caccgtcgca gttgtcgcga gcgcgttcgc accatggggg tgacggccat caccgtggtc 1080

acgctgatgg acggggccgg gcgcatcccc gcc 1113

<210> 4

<211> 1204

<212> DNA

<213> artificial

<220>

<223>TK upstream region of gene homology arm Δs TK-H1

<400> 4

cgtcgtaggc gagcgccacg tcgttcttgg cgatctgcac ctcgcggccc tcgtccgcgg 60

ccgcgaccgc ctcgggcgcc tcctcggcgg cgcgcgccgc gtccgcctcc tcggcggcgc 120

gcgccgtctc ctcgagcacc acgagcgcct cggccacgcg ctccacctgc cgctccagcg 180

gccgcagctg ctcgtccacc tcggcctcga ggcgcgcgcc cgcggccatg gcgttgtcca 240

gcgccgcggc ggccgcgcgg cggcgcgcgt tcgcgtgcgc cagcgcgagg cgcgcgtcga 300

ggccctcgcc gaagcccggg cgggcccaga agcccacggg gaacgggggc gcgatgaagt 360

ggcgcgcgct gcccgggatc gcagcggcct cgaaggcgaa ccacgcgcgg tccatggcgc 420

ggggggacat gggccgcgcg gcggcgcgcg ccgccttatc atccccgctc cccgccgccg 480

cccggcccgg ccccgcgcgc gccgcgatcg cgatcaccgc cgcggcccgg cgacgtactc 540

ggcgaggccg cgcacggtcg cggccatcgc gctcgcgttg ccgcgcgtct gggtgcaggg 600

caggcgcgtc acgtcgagca cgcgcatgct ccgctgggcc acaaacacca gcaggggcac 660

gagcgtgatc tcctcgccgc ccgggggcac ggcggcggcg aggaggcgcg ccgagtcgcg 720

cagctggcac agcccctcgt gccgctgccc gcgcttgctg ggcgtgttga ggttccgggg 780

gaagcggcac gtcttgagct cgatgaggaa gcacaggtgc gggcccgccc ccagccgcac 840

cacgcacacg cagtcggggc ggcgcacccc gaggttgact tcaaaggcca gggtcaagga 900

cgccttctta agcgtctctc ggggaagccc gaagagactc tcgccgtacg cggacgggtc 960

gcgtcgcagg cgttcgtaga agcggttgtg gcagcggatc cccgcccgga agcgcgccgg 1020

gatgcgcatc ctccggatct acctcgacgg cgcctacggc accggcaaga gcaccacggc 1080

ccgggtgatg gcgctcggcg gggcgctgta cgtgcccgag ccgatggcgt actggcgcac 1140

tctgttcgac acggacacgg tggccggtat ttacgatgcg cagacccgga agcagaacgg 1200

cagc 1204

<210> 5

<211> 1216

<212> DNA

<213> artificial

<220>

<223>TK downstream of gene homology arm Δs TK-H2

<400> 5

gctcacggcc ctgcgcaacg tctacgccat gctggtcaac acgtcgcgct acctgagctc 60

ggggcgccgc tggcgcgacg actgggggcg cgcgccgcgc ttcgaccaga ccgtgcgcga 120

ctgcctcgcg ctcaacgagc tctgccgccc gcgcgacgac cccgagctcc aggacaccct 180

cttcggcgcg tacaaggcgc ccgagctctg cgaccggcgc gggcggccgc tcgaggtgca 240

cgcgtgggcg atggacgcgc tcgtggccaa gctgctgccg ctgcgcgtct ccaccgtcga 300

cctggggccc tcgccgcgcg tctgcgccgc ggccgtggcg gcgcaggcgc gcggcatgga 360

ggtgacggag tccgcgtacg gcgaccacat ccggcagtgc gtgtgcgcct tcacgtcgga 420

gatgggggtg tgaccctcgc ccctcccacc cgcgccgcgg ccggatggag accgcgacgg 480

aggcaacgac gacggcgtgg gagggggctc ggggcgcgta taaagccatg tgtatgtcat 540

cccaataaag tttgccgtgc ccgtcaccat gcccgcgtcg tccgtgcgcc tcccgctgcg 600

cctcctgacc ctcgcgggcc tcctggccct cgcgggggcc gccgccctcg cccgcggcgc 660

gccgcagggt gggccgccct cgccgcaggg gggtcccgcg cccaccgcgg cgcccgcgcg 720

cgggcccacc ctgttcgtcc tggacggcga cggctccgcg tggttcgtct tccagctcgg 780

cgggctgggg gcgctcaacg acacgcgcat ccgcgggcac ctgctcggcc ggtacctcgt 840

ctcgtaccag gtggtgcccc cgcccgtctc cgcgtggtac tttgtgcagc gcccgcgcga 900

gcgcccgcgc ctctcggggc cgccctcggg cgcggagctc gtggccttcg acgcgcccgg 960

cgtccggcgc acgtacacca cggcggcggt gtggcccgcg gaggtggccg tcctcgcgga 1020

cgcggaggcg cgctgccccg cggccgtctt caacgtgacg ctgggcgagg ccttcctcgg 1080

cctgcgcgtc gcgctgcgct ccttcctgcc gctggaggtc atcatctccg ccgagcggat 1140

gcgcatgatc gcgcccccgg cgctcggctc ggacctggag ccgccgggcc cgcccgcggg 1200

ccgcttccac gtgtac 1216

Claims

1. the dual-gene missing low virulent strain of pseudorabies virus, is pseudorabies virus LA1206-80 strains, deposit number is：CGMCC No.14329。

2. application of the dual-gene missing low virulent strain of pseudorabies virus described in claim 1 in pseudorabies vaccines are prepared.

3. using the dual-gene missing low virulent strain of pseudorabies virus described in claim 1 as the vaccine of active component.

4. vaccine according to claim 3, it is characterised in that the virus liquid of the dual-gene missing low virulent strain of pseudorabies virus is adopted Prepare with the following method：Pseudorabies virus LA1206-80 strains are inoculated with ST cells, cultivated using the DMEM containing hyclone Base culture 36~48 hours, viral cultures are harvested, centrifuging and taking supernatant is as pseudorabies virus LA1206-80 strains after freeze thawing Virus liquid.

5. vaccine according to claim 4, it is characterised in that the vaccine is live vaccine.

6. a kind of recombinant vector live vaccine, it is characterised in that its active component is double in pseudorabies virus described in claim 1 Insert what is obtained after external source protective antigen gene expression cassette in gene delection low virulent strain genome.