CN1884499A - Recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain vaccine and its uses - Google Patents

Abstract

The invention belongs to animal virogene engineering sphere, and especially to a construction of recombinant-pig encephalitis B-Aujesky's disease gene engineering virus strain, preparation of vaccine and its application. The invention obtains a recombinant-pig encephalitis B-Aujesky's disease gene engineering virus strain Pseudorabies virus WKQ-JE-E001, which is preserved in CCTCC with CCTCC V200509 as its preserved number. The said virus strain deletes major toxicity gene TK of Aujesky's disease and does not produce functional glucoprotein gG; can express E proteantigen of pig encephalitis B virus. The gene schedule strain can stimulate pigs to produce protective immune response to resist pig encephalitis B virus and Aujesky's disease and effectively prevent pig encephalitis B virus and Aujesky's disease affection. The invention also discloses a method of preparing pig encephalitis B-Aujesky's disease gene engineering vaccine with the said gene engineering strain and its application.

Description

A kind of vaccine of recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain and application

Technical field

The invention belongs to the animal virus gene engineering technology field.Be specifically related to a kind of recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain, with the recombinant vaccine and the application of this gene engineered strain preparation.

Background technology

Pseudoabies is by Pseudorabies virus (pseudorabies virus, be called for short PrV) acute infectious disease that causes of multiple domestic animal such as infected pigs, ox, sheep, dog, cat, rabbit, small white mouse, mink, fox and wildlife, pig is the storage person and the circulator (Mettenleiter of this virus, Molecular biology of pseudorabies (Aujeszky ' s disease) virus.Comp Immunol Microbiol Infect Dis, 1991,14:151-163).Other animal except that pig is the form of distributing, and has heating after the morbidity usually, very itches and pattern of fever symptom such as encephalomyelitis, is lethal infection.This disease is eruption and prevalence to pig, and harm mainly causes sow breeding difficulty syndrome and newborn piglet mass mortality.PrV is generally through the pharynx nasalis infection animal, and infecting partial mucous membrane internal breeding, divide a word with a hyphen at the end of a line through lymphokinesis or nerve fiber then, it is generally acknowledged that virus arrives brain and causes the nervus centralis dysfunction through nerve pathway, or, set up latent infection (Mettenleiter in the trigeminal nerve at olfactory bulb, Immunobiology of pseudorabies (Aujeszky ' s disease), Vet Immunol Immunopathol, 1996,54:221-229).

The genome of Pseudorabies virus is the wire double-stranded DNA, be about 150kbp, G+C content is up to 74%, whole genome 70-100 the albumen of encoding at least, wherein thymidine kinase (Thymidine Kinase, TK) gene is positioned at UL district, total length 963bp, 320 amino acid of encoding, catalytic deoxidation thymidine phosphorylation.The TK gene is relevant with the virulence of PrV, it is the main virulence gene of Pseudorabies virus, relevant (Kit S etc. with the latent infection of virus, Attenuated properties of thymidinekinase-negative deletion mutant of pseudorabies virus.Am J Vet Res, 1985,46:1359-1367; Nunberg etc., Identification of the thymidine kinase gene of felineherpesvirus:use of degenerate oligonucleotides in the polymerase chain reaction toisolate herpesvirus gene homologs, J Virol, 1989,63:3240-3249).Except the TK gene, the gene relevant with the Pseudorabies virus virulence also has gG, gE etc.TK, gE and gI gene all are that virus multiplication institute is nonessential, disappearance virus strain and the same can normally propagation of wild poison, and do not exist virulence to return strong danger (Thomsen, PRV as a livevector for expression of foreign gene, Gene, 1987,57:261-265).The mutated viruses strain has good immunogenicity; can produce protective immunological reaction behind the inoculation animal; but do not produce the antibody of anti-gE; therefore can be used as a sign distinguishing immune animal and wild virus infection animal; help setting up the swinery that no pseudoabies infects, and finally provide useful instrument for eradicating this disease.Hua Zhong Agriculture University is at the strong malicious Hubei Province A strain (Chen Huanchun etc. of Pseudorabies virus of autonomous isolation identification, the isolation identification of porcine pseudorabies virus Hubei Province A strain, journal of animal science and veterinary medicine, 1998, on basis 29:151-156), with main virulence TK gene, nonessential glycoprotein gG or gE, gI genetically deficient, elimination (Liu Zhengfei etc., pseudorabies virus Ea strain TK that a series of recombination mutation strain is used for pseudoabies have been made up by the DNA recombinant technology ^-/ gE ^-The structure of/gp63-mutant strain and biological characteristic research thereof, microorganism journal, 2002,42:370-374; Liu Zhengfei etc., pseudorabies virus Hubei Province A strain TK ^-/ gE ^-/ gI ^-The security of gene-deleted vaccine and protection research, journal of animal science and veterinary medicine, 2004,35:70-73; Xu Xiao cuckoo etc., pseudorabies virus TK ^-/ gG ^-The structure and the biological characteristic research thereof of disappearance strain, the biotechnology journal, 2004,20:532-535).

Encephalitis B (Japanese encephalitis is called for short JE) is called for short encephalitis, is the carapuru virus disease that a kind of people and animal suffer from altogether.This disease is found in Japan the earliest.This disease just had Preliminary study clinically in 1871, a kind of special transmissible disease is thought by the time side of being very popular to nineteen twenty-four.This disease is mainly popular in summer and autumn, once is called " summer encephalitis ".In order to be different from the lethargic sleep encephalitis (encephalitis lethargica) that once took place in the winter time, Japanese scholar (nineteen twenty-eight) with winter the popular encephalitis be called encephalitis A, autumn in summer popular encephalitis is called encephalitis B.The many countries and regions in the Far East and South East Asia mainly take place and are popular in this disease.

Encephalitis B endangers huge to the mankind, in China, Japan and other West Pacific Ocean countries, encephalitis B is one of modal vector borne diseases of human central nervous system (CNS), and it is to cause a dead and disabled major cause in the whole Asia that comprises the Indian subcontinent.

Encephalitis B case fatality rate height, average out to 20%, external some places are up to 50%-60%, the whole world number of dying of illness in every year surpasses 10,000 people (Burke etc., Intense transmission of Japanese encephalitis virus to pigs in a regionfree of epedemic encephalitis.Southest Asian J Trop Med Public Health, 1985,16 (2): 199-206; Vaughn etc., The epidemiology of Japanese encephalitis:prospects forprevention.Epidemiol Rev, 1992,14:197-221).Its sequela is serious, the healing person of about 57%-68% has the neuroscience sequela, wherein severe patient accounts for 18% (comprise nonvoluntary quadruped locomotion, epilepsy, motor aphasia, paralysis, blind etc.), slight motion and mental function obstacle are difficult to when generally checking in early days find, great majority could be found in the tracking sight in 5 years is looked into, find that through following up a case by regular visits to about half patient has memory defects, 3/4ths have ataxia and the impaired (Yu Yongxin of mental capabilities, the international encephalitis B control strategy of provincialism meeting brief introduction, the Chinese biological goods are learned magazine, 1996,9:46-48).So Duo sequela patient is that patient, family, society bring serious harm and burden.

Related data shows (Song Dingbo etc., popular and the prevention and control of Japanese encephalitis. Chinese Frontier Sanitary Quarantine magazine, 2005,28:348-350), entered since the nineties, encephalitis B popular scale such as Singapore, Japan, Korea and number of times are reducing year by year, in China and some other Asian countries, except some higher areas of immunization rate, there is encephalitis B popular always.According to incompletely statistics, global annual encephalitis B case be 5-10 ten thousand (Yu Yongxin, the international encephalitis B control strategy of provincialism meeting brief introduction. the Chinese biological goods are learned magazine, 1996,9:46-48).As seen, encephalitis B is still threatening human beings'health in various places.

A variety of animals such as pig, horse, ox, goat, sheep, monkey and poultry be the easy infection encephalitis b virus very, and other is as rabbit, rat, dove, dog, duck, wild fowl and reptiles also susceptible, and mouse and some lizard can be by experimental infections.Its clinical symptom is different and different with the infected animal kind.Ox, sheep, and poultry mostly be inapparent infection, serious encephalitis takes place in the Marko.Pig mainly shows as: latent period 3d-4d, the high heat of ill cub is delaied, spirit is depressed, walking is staggered, last body paralysis and dead; Growing and fattening pigs continue high heat; Pregnant sow mainly shows as miscarriage, the stillborn foetus that output differs in size, and monster, mummy tire and weak young does not generally influence the breeding next time post-abortion; One-sided or the both sides property testis enlargement of boar, local pyrexia has pain, and after a couple of days, testis swelling is disappeared, atrophy hardening gradually.Clinical symptom appears in susceptible piglet once in a while, not necessarily shows clinical symptom after grow up pig or conceived pig infect encephalitis B.Yet after conceived susceptible sow was infected, in various degree unusual can appear in final fetus.

In the experimental infection that the farrowing sow to susceptible carries out, parent does not have clinical symptom, but the intrauterine infection that has caused fetus, farrowing is unusual, stillborn foetus, the mummy fetus that different quantities differs in size occur and neurological symptom, subcutaneous dropsy, hydrocephalic weak young are arranged.Cut open inspection visible sow uterine mucosa hyperemia, hemorrhage, oedema and erosion.The boar testitis causes boar sterile, summer boar sterile may be with to infect encephalitis B relevant.Oedema appears in the boar testis that infects encephalitis b virus, parenchyma of testis hyperemia, and have wedge-like or mottled hemorrhage and necrosis region, epididymis hardening, sexual desire to weaken, and discharge virus by seminal fluid.In the seminal fluid of morbidity boar, sperm sum and great-hearted sperm count obviously reduce, and with a large amount of abnormal spermiums.In most of cases, this damage is temporary, can recover fully at last, but also see once in a while have serious case cause permanent sterile (Zheng family is third-class, the Japanese B encephalitis of pig. raise pigs, 2005,6:37-38).

The main financial loss that this disease causes is that breeding difficulty takes place farrowing sow, the pig industry as the livestock industry mainstay industry is continued volume increase cause huge harm.Vaccine inoculation is reliable tools and the effective means as specificity prevention encephalitis B.At present, Vaccinum Encephalitis B mainly contains inactivated vaccine and these two kinds of conventional vaccines of attenuated vaccine, though the certain effect of performance in the anti-system of encephalitis B of these two kinds of vaccines exists some shortcomings.Inactivated Japanese Encephalitis Vaccine have production cost height, injected dose big, need weak points such as multiple injection, effect instability and immunizing power are not lasting, mouse brain deactivation vaccine also contains more cerebral tissue composition in addition, and serious allergic encephalomyelitis easily takes place in the inoculation back.And the relevant danger that inevitably exists the poison that looses of inactivated vaccine production process with live virus.A little less than the causing of the weak poison of encephalitis B is to be caused by single coding mutation after all, and the possibility that its virulence reverses can not be got rid of, so the security of encephalitis B attenuated live vaccines is troubling always.

Except above-mentioned conventional vaccine, people are seeking encephalitis B recombinant vaccine such as subunit vaccine, nucleic acid vaccine, live vector vaccines such as poxvirus, adenovirus.But still there is some deficiency in these vaccines, be difficult to be accepted by people, involve great expense as subunit vaccine, nucleic acid vaccine can produce protection antibody by the induction of immunity animal, its validity is firmly established, but because the theoretical crisis (whether can be incorporated in the host cell gene group) that nucleic acid vaccine faced is not resolved, its possibility that enters clinical application at no distant date is very little; Though the vaccine of live vector such as poxvirus, adenovirus has certain immune effect, have certain side effect, this has limited this class vaccine application of future in clinical.Therefore, press for to develop and have safe, efficient, cheap novel encephalitis B recombinant vaccine.

Summary of the invention

The objective of the invention is to overcome the defective that prior art exists, obtain the better and stronger recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain of security of a kind of immunogenicity;

Second purpose of the present invention is to utilize this recombination engineering strain to prepare pig japanese b encephalitis-pseudoabies recombinant vaccine;

The 3rd purpose of the present invention is the application of recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain on preparation pig japanese b encephalitis-pseudoabies recombinant vaccine.

The present invention is achieved through the following technical solutions:

A kind of recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain Pseudorabies virus WKQ-JE-E001 is deposited in CCTCC, deposit number: CCTCC-V200603.

The main virulence gene TK of described recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain disappearance Pseudorabies virus does not produce functional glycoprotein gG, and can correctly express pig japanese b encephalitis virus E gene.

A kind of recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain, the TK that described gene engineered strain relates to ^-/ gG ^-/ LacZ ⁺Parent plant is the exquisite weak genetically engineered mutant strain of a strain people, it is derived from applicant's PRV (Pseudorabies virus) Hubei Province A strain of open report before the applying date, described Hubei Province A strain strain is separated, is identified by Hua Zhong Agriculture University animal virus research department, be that the titre height is bred in a strain on cell culture, the virulent strain that immunogenicity is strong is (referring to Chen Huanchun etc., the isolation identification of porcine pseudorabies virus Hubei Province A strain, journal of animal science and veterinary medicine, 1998,29:151-156).Its main virulence gene TK of described recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain has lacked 205bp, causes virulence to reduce greatly; Big fragment takes place and inserts disappearance in the gG gene, causes not producing glycoprotein gG, can duplicate in noble cells, but duplicating in undifferentiated neurocyte is prevented from or limits, even latent infection takes place in the inoculation back, also be difficult for being activated, thereby have very high security.

Basic construction method of the present invention is: (be called for short JEV) main immunogenic gene E gene is inserted into Pseudorabies virus TK down together with pig japanese b encephalitis to utilize genetic engineering technique ^-/ gG ^-/ LacZ ⁺In the genome of vaccine strain, be located at the downstream of gG promotor, express the back a few amino acids formation fusion rotein initial with gG albumen.Because the E gene that inserts itself has termination codon and poly (A) sequence, and do not merge with gG albumen end sequence, so the proteic biological characteristics of E of expressing does not change, gG albumen still can not get expressing simultaneously.Recombinant strain by a large amount of biological experiment digital proof the present invention preparation can be used for preparing the application on pig japanese b encephalitis-pseudoabies recombinant vaccine.

Major advantage of the present invention is:

1, the used solid support material of the present invention is Pseudorabies virus Hubei Province A strain TK ^-/ gG ^-/ LacZ ⁺The genetically engineered attenuated vaccine strain; this vaccine strain is not only fool proof effectively to growing and fattening pigs, piglet; even it is also fool proof to newborn piglet; can produce strong protection; and can be used for the leading immunity of piglet; have excellent prevention and result of treatment, this just provides wide prospect for it being developed to a kind of good viral lived vaccine carrier.

2, the gG albumen of pseudoabies vector virus can not be expressed, therefore, the vaccine of this gene engineered strain preparation can be used as the marker vaccine (animal of marker vaccine immunity of eradicating Pseudorabies virus and pig japanese b encephalitis virus, can utilize corresponding differential diagnosis method, distinguish mutually) with the animal of wild virus infection.

3, recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain of the present invention does not contain external source LacZ gene.

Description of drawings

Fig. 1: be the physical map of expressing encephalitis b virus E transgenosis plasmid pgG-E among the present invention.

Fig. 2: be pgG-E transferring plasmid qualification result of the present invention.

M:DNA marker among the figure (DL 15000) 1:pgG-E/BamHI+EcoRI 2:pgG-E/BamHI

Fig. 3: be the schema that pgG-E transferring plasmid of the present invention makes up.

Fig. 4: be the recombinant pseudorabies virus strain TK that expresses encephalitis b virus E gene among the present invention ^-/ gG ^-/ E ⁺The electrophoretogram that detects of PCR.

Among the figure 1: plasmid pgG-E contrasts 2-5: recombinant pseudorabies virus strain TK ^-/ gG ^-/ E ⁺6: parent plant PrV TK ^-/ gG ^-/ LacZ ⁺Contrast 7:IBRS-2 cell contrast 8:H ₂The contrast of O negative control M:DL2000DNA molecular weight

Fig. 5: be the recombinant pseudorabies virus strain TK that expresses encephalitis b virus E gene among the present invention ^-/ gG ^-/ E ⁺Synoptic diagram.

Fig. 6: be the expression encephalitis b virus E gene recombination pseudoabies strain TK among the present invention ^-/ gG ^-/ E ⁺The Southern-blotting analytical results.

1:TK among the figure ^-/ gG ^-/ LacZ ⁺/ BamHI+EcoRI 2:TK ^-/ gG ^-/ E ⁺/ KpnI 3:TK ^-/ gG ^-/ E ⁺/ BamHI+EcoRI4:TK ^-/ gG ^-/ LacZ ⁺/ KpnI

Fig. 7: be the expression encephalitis b virus E gene recombination pseudoabies strain TK among the present invention ^-/ gG ^-/ E ⁺The Western-blotting analytical results.

Among the figure 1: infect TK ^-/ gG ^-/ E ⁺The IBRS-2 cell 2 of virus strain: infect TK ^-/ gG ^-/ LacZ ⁺The IBRS-2 cell M of virus strain: protein Marker

Embodiment

The present invention is further illustrated below in conjunction with Figure of description

1, design of primers (being used for gene clone and Molecular Detection)

According to a pair of primer amplification E gene of having reported of JEV SA14-14-2 strain sequence (the GenBank accession number is AF315119) design, the amplified fragments size is 1572bp, and two ends are designed BamHI and EcoRI restriction enzyme site respectively.Above-mentioned primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.Primer sequence is as follows:

Pe1：5’-GCGGATCCATGCTTGGCAGTAACA-3’

Pe2：5’-GCGAATTCTAAAGCATGCACATTGGTCGC-3’

2. the clone of encephalitis b virus E gene

Go up inoculation JEV SA14-14-2 strain (Wang Zhiwei etc. at the Chinese hamster kidney passage cell (BHK-21) that covers with individual layer; domestic and international 3 kinds of Vaccinum Encephalitidis Epidemicaes in the intravital protection test of mouse relatively; the Chinese public health journal; 1999; 18:69-71); connecing poison back generation 80% left and right sides cytopathy, centrifugal collecting infecting cell extracts test kit (RNA-Solv according to the RNA of OMEGA Bio-Tek company (U.S.) ^Reagent) specification sheets extracts total RNA of cells infected as the RT-PCR template.Extracting the rifle head, the transfer pipet that relate in the RNA process all handles through 0.5% DEPC water (volume/volume): promptly in rifle head or transfer pipet the DEPC water 0.5% 37 ℃ soak 24-48h after, 121 ℃ of autoclaving 30min preserve standby.Concrete RNA extraction step is: the BHK-21 cell suspension and the 1mL RNA-Solv that draw the above-mentioned infection JEV of 200 μ L SA14-14-2 virus ^Reagent mixes mutually, behind the room temperature effect 5min, trichloromethane (chloroform) the vortex mixing that adds 200 μ L again, behind the strict ice bath 10min, the centrifugal 5min of 12000rpm, draw 80% (guaranteeing not to be drawn onto egg white layer) of upper phase, be transferred in the new transfer pipet, add isopyknic Virahol mixing, room temperature is placed 5-10min, the centrifugal 10min of 12000rpm, the careful suction abandoned liquid, precipitate with after 75% the dehydrated alcohol rinsing once, the DEPC water dissolution of air-dry adding 20 μ L ,-70 ℃ of preservations are standby.

The amplification reaction system of E gene is: 10 * reaction buffer, 5 μ L, 25mM MgCL ₂10 μ L, 10mM dNTPs 5 μ L, RNA enzyme inhibitors 1 μ L, AMV ThermoScript II XL 1 μ L, (RNA of the equal OMEGA Bio-Tek of above reaction reagent company (U.S.) extracts test kit (RNA-Solv to AMV-optimized Taq enzyme 1 μ L ^Reagent) provide), primer Pe1 (final concentration is 10pmol/L) 2 μ L, primer Pe2 (final concentration is 10pmol/L) 2 μ L, the RNA template 5 μ L of said extracted add DEPC water to 50 μ L.By 50 ℃ of effects 30 minutes, 94 ℃ acted on 2 minutes; 94 ℃ act on 40 seconds, annealed 30 seconds for 58 ℃ then, 72 ℃ were extended 2 minutes, totally 35 circulations, last 72 ℃ of reaction conditionss that extended 10 minutes increase on the PCR instrument, amplification PCR products is through 0.8% agarose gel electrophoresis analysis, in pMD-18 carrier (available from the precious biotechnology in Dalian company limited), and send Dalian precious biotechnology company limited to carry out the mensuration of exogenous gene sequence simultaneously the E gene clone that obtains.Referring to shown in Figure 1.

3.pgG-E the structure of transferring plasmid

With BamHI and EcoRI respectively enzyme cut E gene PCR amplified production and the carrier pgG-uni (Fang Liurong etc. that size is 159lbp, the structure of the general transfer vector of pseudorabies virus gG genetically deficient, Hua Zhong Agriculture University's journal, 2001,20:306-309), reclaim E gene and carrier pgG-uni, use T then ₄DNA ligase connects, and 16 ℃ of water-baths are spent the night, and transforms DH5 α competence bacterium, and bacterium is chosen in 37 ℃ of cultivations, will connect product then and transform DH _{5 α}Intestinal bacteria, a small amount of prepares plasmid, enzyme is cut evaluation, thereby obtains transferring plasmid pgG-E.Its physical map is seen Fig. 1.Qualification result confirms the correct (see figure 2) of transferring plasmid pgG-E of structure, and transferring plasmid pgG-E makes up flow process as shown in Figure 3.

4. recombinant virus TK ^-/ gG ^-/ E ⁺Structure

Propagation PrV TK on the IBRS-2 cell ^-/ gG ^-/ LacZ ⁺Parent plant, extract its genomic dna (extracting method is with reference to Wang Liu etc., the molecular cloning of human cytomegalic inclusion disease virus and the preparation of specificity DNA probing needle thereof, biotechnology, 1994,4:33-35).Because PrV TK ^-/ gG ^-/ LacZ ⁺Genomic dna in only have an EcoRI restriction enzyme site, and be arranged in the gG gene.With EcoRI with PrV TK ^-/ gG ^-/ LacZ ⁺The genomic dna enzyme be cut into two sections, (method is with reference to Zhou Fuchun etc., pseudorabies virus Hubei Province A strain gG to use liposome-mediated rotaring dyeing technology ^-/ LacZ ⁺The structure of mutant strain, journal of animal science and veterinary medicine, 2001,32:129-133) with itself and pgG-E transfer vector cotransfection IBRS-2 cell, by homologous recombination takes place the E gene is transferred in the PrV genome gG gene, has displaced the LacZ gene simultaneously, receive poison after waiting to produce cytopathy.Be inoculated in the IBRS-2 cell with receiving the malicious viral liquid that obtains, keep liquid with the DMEM that contains 1% low melting-point agarose (available from U.S. GIBCO company product) and (contain 5% new-born calf serum, this bovine serum is available from the biological company limited of Chinese Hangzhou folium ilicis chinensis) cover, in 37 ℃ of 5% CO ₂Condition under cultivate, when just cytopathy occurring, toluylene red dyeing 1h with 0.01%, because sick cell can not be painted, and normal cell is dyed redness, thereby the round point shape plaque appears, the single plaque of picking is inoculated IBRS-2 cell enlarged culturing, utilizes E gene PCR detection method (Xu Gaoyuan etc., the clone of encephalitis b virus SA14-14-2 strain E gene of JEV, order-checking and expression., China Amphixenosis magazine, 2004,20:22-25) identify positive recombinant virus (PCR the results are shown in Figure 4), so carry out purifying three times, utilize PCR detection method (Liu Zhengfei etc., the pseudorabies virus Ea strain TK of LacZ gene at last ^-/ gE ^-/ gp63 ^-The structure of mutant strain and biological characteristic research thereof, the microorganism journal, 2002,42:370-374) can not detect PrV TK ^-/ gG ^-/ LacZ ⁺The existence of strain, thus determine only to contain recombinant pseudorabies virus TK in the viral liquid ^-/ gG ^-/ E ⁺

5. recombinant virus (TK ^-/ gG ^-/ E ⁺) biological characteristics and evaluation thereof

With reference to Sa Mubulu I, not Ritchie E and Manny A Disi T. are outstanding, second edition, Jin Dongyan etc. translate the school, " molecular cloning experiment guide ", Science Press, Beijing, 1998) method in carries out Southern-blotting, SDS-PAGE and Western-blotting identifies.

Southern-blotting identifies: extract recombinant pseudorabies virus TK ^-/ gG ^-/ E ⁺The genomic dna of strain (reference: Wang Liu etc., the molecular cloning of human cytomegalic inclusion disease virus and the preparation of specificity DNA probing needle thereof, biotechnology, 1994,4:33-35) cut with BamI+EcoRI, KpnI enzyme after, at 30 volts of electrophoresis on 0.3% the sepharose after 6 hours, (Sa Mubulu I according to a conventional method, not Ritchie E and Manny A Disi T. are outstanding, " molecular cloning experiment guide " second edition, and Jin Dongyan etc. translate the school, Science Press, Beijing, 1998) transfer on the cellulose acetate membrane, standby behind 80 ℃ of roasting films.Pcr amplification E gene fragment, after adopting DNA to reclaim test kit (giving birth to worker bio-engineering corporation) recovery available from Shanghai, with digoxigenin labeled and detection kit (available from U.S. Roch company product) mark (the test kit specification sheets operation that concrete grammar provides by Roch company), dna probe as the E gene places-20 ℃ of preservations standby.With the dna probe of E gene and standby cellulose acetate membrane hybridization, the result as shown in Figure 6 at last.Test-results shows, as a result the TK that cuts of BamHI+EcoRI, KpnI enzyme ^-/ gG ^-/ E ⁺Genomic dna locates to occur a specific hybrid belt respectively about 1.6kb and 5.4kb, size conforms to expection.And TK ^-/ gG ^-/ LacZ ⁺No matter genomic dna is to cut or cut with the KpnI enzyme with the BamHI+EcoRI enzyme all not have any hybrid belt and occur.Confirm that the E gene has been inserted in the genome of parent plant.

SDS-PAGE and Western-blotting identify: with recombinant pseudorabies virus TK ^-/ gG ^-/ E ⁺Strain is inoculated in the IBRS-2 cell that just grows up to individual layer, collects sick cell after 16 hours.According to a conventional method (Sa Mubulu I, not Ritchie E and Manny A Disi T. are outstanding, " molecular cloning experiment guide ", second edition, Jin Dongyan etc. translate the school, Science Press, Beijing, 1998) prepare sample, carry out SDS-PAGE and Western-blotting, the result is as shown in Figure 7.Analyze through SDS-PAGE, infect TK ^-/ gG ^-/ E ⁺The IBRS-2 cell swimming lane of strain, find a specificity band at about 53kD place, Western-blotting analyze to find its can with pig japanese b encephalitis virus-positive sero-reaction, a specific hybrid band appears, then do not have in two contrast swimming lanes, show that the pig japanese b encephalitis viral protein has obtained expression.

Recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain Pseudorabies virus WKQ-JE-E001 of the present invention delivers Chinese typical culture collection center (CCTCC) preservation on June 9th, 2006, and its deposit number is CCTCC-V200603.

Recombinant pseudorabies virus strain TK ^-/ gG ^-/ E ⁺Be the present invention constructed with the dual-gene disappearance strain of Pseudorabies virus TK ^-/ gG ^-/ LacZ ⁺Be virus vector, the main immunogenic gene E gene of pig japanese b encephalitis virus is inserted into the dual-gene disappearance strain of Pseudorabies virus TK ^-/ gG ^-/ LacZ ⁺Genome in, the artificial constructed divalence attenuated vaccine strain that obtains.Its genetic stability and biological characteristics and the A strain of parent's strain Hubei Province do not have difference.It is double-stranded DNA that recombinant virus has cyst membrane, nucleic acid, and the virion diameter is 120-180nm, to ether, chloroform and trypsinase sensitivity, and can complete inactivation in 56 ℃ of 30min.TCID on the IBRS-2 cell ₅₀Still can reach 10 ^-7.5/ mL.Recombinant virus was uploaded for 30 generations at the IBRS-2 cell continuously, and its propagation titre does not have obvious variation, expression pig japanese b encephalitis virus E protein that can be stable.

This recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain need be gone up propagation and detect survival condition at pig kidney subculture cell (IBRS-2), the substratum of IBRS-2 cell is Dulbecco ' the s Modified EagleMedium (DMEM) (available from U.S. GIBCO company product) that contains 10% calf serum, the pH scope is 6.8-7.2, in 37 ℃ of cultivations.Can adopt routine to freeze and/or conventional lyophil preservation.

6, pig japanese b encephalitis-pseudoabies recombinant vaccine (TK ^-/ gG ^-/ E ⁺) preparation

With the recombinant pseudorabies virus TK that obtains ^-/ gG ^-/ E ⁺Identify, every pickup kind IBRS-2 cell, utilize the E gene of JEV to carry out PCR detection (Xu Gaoyuan etc., the clone of encephalitis b virus SA14-14-2 strain E gene, order-checking and expression, China Amphixenosis magazine, 2004, the 20:22-25) genetic stability of the foreign gene of evaluation recombinant virus, back its E gene of discovery that goes down to posterity for 30 times still correctly is positioned among the gG site genetic stability.And detecting foreign protein by Western-blotting can stably express, and has good biologic activity.The propagation titre of this recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain on pig kidney passage cell reaches 10 ^7.6TCID ₅₀, through screening, adopting the easy chick embryo fibroblast propagation vaccine virus of preparation, its propagation titre reaches 10 ^7.0TCID ₅₀With vaccine kind poison by 1/10 volume ratio inoculated into chick embryo inoblast; collect qualified viral liquid after the cytopathy; in viral liquid: gelatin protective material (volume/volume) is that 7: 1 ratio adds the gelatin protective material (this gelatin protective material compound method is: in every 100ml deionized water with sucrose 40g; gelatin 8g; after fully melting; preservation is standby after putting 121 ℃ of 30min that sterilize down); in sterilization freeze-drying bottle, press the packing of 2.5mL/ bottle; put freeze-drying in the freeze drier; freeze-drying 36-40h rear pressing cover, warp is at IBRS-2 cell detection TCID ₅₀Reach 10 ^5.6It is standby behind the/0.1mL, to put-20 ℃ of preservations.

7, pig japanese b encephalitis-pseudoabies recombinant vaccine (TK ^-/ gG ^-/ E ⁺) safety evaluation

Recombinant vaccine (TK with the present invention's preparation ^-/ gG ^-/ E ⁺) 30 of the Balb/c small white mouse of inoculation about 18g, 100 μ l inject in every small white mouse both sides hind leg foot pad portion, inoculate 100 μ l (10 altogether ^6.0TCID ₅₀), establish vector virus vaccine (TK simultaneously ^-/ gG ^-/ LacZ ⁺), the A strain of Pseudorabies virus Hubei Province and non-immune group make blank, observes the clinical symptom of small white mouse, observed continuously 14 days.Found that the recombinant vaccine (TK of the present invention's preparation ^-/ gG ^-/ E ⁺) and vector virus vaccine (TK ^-/ gG ^-/ LacZ ⁺) small white mouse of inoculation does not have any ANOMALOUS VARIATIONS, but all death in 1 week of Hubei Province A strain infecting mouse show that the recombinant vaccine that the present invention prepares is safe (table 1) to small white mouse.

Recombinant vaccine strain (the TK of table 1 the present invention preparation ^-/ gG ^-/ E ⁺) to the safety evaluation of Balb/C small white mouse

The test group	Number of animals (only)	Death toll (only)	Mean time to death (hour)
				TK -/gG -/LacZ +(vector virus) TK -/gG -/E +(the present invention) Hubei Province A street strain infected group (contrast 1) blank group (contrast 2)	30 30 30 30	0 0 30 0	0 0 113.5±36.8 0

Recombinant vaccine with the present invention's preparation) (TK ^-/ gG ^-/ E ⁺) (contain 10 by every pig injection 2mL ⁶TCID ₅₀Virus quantity) inoculation 25 ages in days piglet, part is inoculated the exothermic reaction that a slight property crossed appears in piglet, recover normal two days later, the spiritual appetite of piglet of recombinant vaccine of injecting the present invention preparation during this period is normal, no abnormality seen changes, and can detect the Pseudorabies virus neutralizing antibody.The piglet (promptly contrasting 1) of PrV Hubei Province A virulent strain inoculation all presented exothermic reaction in second day, continues a week, and a death is arranged, and cut open inspection and find to have typical pseudoabies pathological change, and control group 2 had not both had fervescence, did not have unusual clinical manifestation yet.(contain 10 by every pig injection 2mL ⁶TCID ₅₀Virus quantity) inoculate TK of the present invention ^-/ gG ^-/ E ⁺Vaccine and nonvaccinated pregnant sow, the nest litter size is suitable substantially, stillborn foetus, mummy tire etc. all do not occur, confirms that the recombinant vaccine of the present invention's preparation is safe to pregnant sow.

8. pig japanese b encephalitis-pseudoabies recombinant vaccine (TK ^-/ gG ^-/ E ⁺) detect at the intravital immune efficacy of mouse

1) Pseudorabies virus is attacked the protectiveness test of Balb/C small white mouse:

Respectively with containing 10 ^5.0TCID ₅₀Recombinant vaccine (the TK of the present invention's preparation of virus titer ^-/ gG ^-/ E ⁺) and vector virus vaccine (TK ^-/ gG ^-/ LacZ ⁺) 0.1mL is through 30 of the inoculation Balb/c of hind leg foot pad portion small white mouses, uses 100LD after 7 days ₅₀Hubei Province A street strain attack poison.Establish JEV SA14-14-2 vaccine strain (Wang Zhiwei etc. simultaneously; domestic and international 3 kinds of Vaccinum Encephalitidis Epidemicaes in the intravital protection test of mouse relatively; the Chinese public health journal; 1999; 18:69-71) immune group and non-immune group are made negative control; observed the vaccine and the vector virus vaccine (TK of the present invention's preparation 21 days ^-/ gG ^-/ LacZ ⁺) the immune group small white mouse all is protected, and none existence of the small white mouse of control group.Presentation of results the insertion of encephalitis b virus E gene do not influence the immune effect (as table 2) of carrier bacterin to Pseudorabies virus.

Recombinant vaccine strain (the TK of table 2 the present invention preparation ^-/ gG ^-/ E ⁺) protectiveness of Pseudorabies virus being attacked the Balb/C small white mouse

The test group	Number of animals (only)	Attacked poison back 21 days		Protection ratio	Mean time to death (hour)
						Death toll	The survival number
		TK -/gG -/LacZ +(vector virus) TK -/gG -/E +(the present invention) JEV SA14-14-2 (contrast 1) blank group (contrast 2)	30 30 30 30					0 0 30 30	30 30 0 0	100％ 100％ 0％ 0％	0 0 116±31.8 112±26.5

2) immune programme for children of mouse:

Select body weight be Balb/c mouse about 18g as the immune efficacy evaluation, be divided into 4 groups according to test requirements document, be respectively the recombinant vaccine immune group (TK of the present invention's preparation ^-/ gG ^-/ E ⁺), vector virus vaccine (TK ^-/ gG ^-/ LacZ ⁺), JEVSA14-14-2 vaccine strain immune group and non-immune blank group, 30 every group, immunization route is that back leg intramuscular injection 100 μ l (contain 10 ^5.0TCID ₅₀Virus quantity) viral liquid or DMEM substratum (available from U.S. GIBCO company), (method is with reference to Xu etc. to exempt to detect in the 4th and 8 weeks of back the humoral immunity level of JEV respectively at head, Construction of recombinant pseudorabiesvirus expressing NS1 protein of Japanese encephalitis (SA14-14-2) virus and itssafety and immunogenicity.Vaccine, 2004,22:1846-1853), and the specific killing BHK-21 cell efficient that infects JEV is that (method is with reference to Xu etc. for cytotoxic T cell specific killing activity (CTL), Constructionof recombinant pseudorabies virus expressing NS1 protein of Japanese encephalitis (SA14-14-2) virus and its safety and immunogenicity.Vaccine, 2004,22:1846-1853).

3) immune mouse humoral immunization and cellular immunization detect

Take a blood sample first and after head exempts from, carried out in 4 weeks, take a blood sample through tail vein negative pressure, separation of serum, (method is with reference to Xu etc. for the ELISA antibody of the anti-JEV of detection specificity, Construction of recombinant pseudorabies virus expressing NS1protein of Japanese encephalitis (SA14-14-2) virus and its safety andimmunogenicity.Vaccine, 2004,22:1846-1853).Blood sampling is for the second time exempted from the back at head and was carried out in 8 weeks, directly through eyeball bloodletting separation of serum, (method is with reference to Xu etc. to detect ELISA antibody, Construction of recombinantpseudorabies virus expressing NS1 protein of Japanese encephalitis (SA14-14-2) virusand its safety and immunogenicity.Vaccine, 2004,22:1846-1853).The results are shown in Table 3.As can be seen from Table 3, head exempts from 4 weeks of back, the vaccine strain and the SA14-14-2 vaccine strain immune group antibody male rotary rate of the present invention's preparation all reach 100%, the vaccine strain of the present invention's preparation induces the antibody titer of generation between 1: 40～1: 80, the SA14-14-2 vaccine strain induces the antibody titer of generation between 1: 40～1: 160, and the vector virus (TK that carries out synchronously ^-/ gG ^-/ LacZ ⁺) and blank all negative.Two exempt from 4 weeks of back (being that head exempts from 8 weeks of back), the vaccine strain of the present invention's preparation and the antibody of SA14-14-2 vaccine strain immune group all have rising to a certain degree, the antibody titer that the two produced is suitable substantially, its antibody titer between 1: 40～1: 320, vector virus (TK ^-/ gG ^-/ LacZ ⁺) and the antibody test of blank group still negative.The above results can induce body to produce the humoral immunoresponse(HI) of specific anti-JEV after showing the recombinant vaccine immune mouse that the present invention prepares.

Recombinant vaccine strain (the TK of table 3 the present invention preparation ^-/ gG ^-/ E ⁺) immune mouse JEV Serum Antibody Detection (ELISA method)

Group	Immunizing dose (TCID 50)	Head exempts from 4 weeks of back				Head exempts from 8 weeks of back
										＜1∶40	1∶40	1∶80	1∶160	＜1∶80	1∶80	1∶160	1∶320
		JEV SA 14-14-2 TK -/gG -/E + TK -/gG -/LacZ +The blank group	By specification carries out (0.2mL) 10 5.0 10 5.0 DMEM	0/30 a 0/30 30/30 30/30	13/30 16/30 0/30 0/30	13/30 11/30 0/30 0/30	4/30 3/30 0/30 0/30	3/30 4/30 30/30 30/30	9/30 8/30 0/30 0/30									12/30 13/30 0/30 0/30	6/30 5/30 0/30 0/30

^aWhat represent is the quantity/total immune quantity that reaches certain antibody titer mouse

Head exempts from 8 weeks of back and puts to death mouse, the aseptic spleen of getting, the preparation splenic lymphocyte, detecting its cytotoxic T cell specific killing (CTL), active (method is with reference to Xu etc., Construction of recombinant pseudorabies virus expressingNS1 protein of Japanese encephalitis (SA14-14-2) virus and its safety andimmunogenicity.Vaccine, 2004,22:1846-1853).The result is as shown in table 4.Vector virus (TK ^-/ gG ^-/ LacZ ⁺) and blank group CTL activity be respectively 11.20% and 8.68%.The vaccine strain of the present invention's preparation and the CTL activity of SA14-14-2 vaccine strain immune group are 54.57% and 61.30%.Analyze the vaccine strain immune group and the vector virus (TK of the present invention's preparation by statistics ^-/ gG ^-/ LacZ ⁺) compare with negative control group, difference is extremely significantly (P＜0.01) all, and not remarkable with SA14-14-2 vaccine strain immune group difference.The result shows that the recombinant vaccine that the present invention prepares can produce the specific CTL activity of JEV by inducing mouse.

Vaccine strain (the TK of table 4 the present invention preparation ^-/ gG ^-/ E ⁺) immune mouse brings out JEV Specific CTL Cells cytotoxic activity (X ± SD) (head exempts from 8 weeks of back)

Group	Number of animals (only)	CTL cytotoxic activity (%)
			JEV SA14-14-2 (contrast 1) TK -/gG -/E + TK -/gG -/LacZ +(vector virus) blank group (contrast 2)	30 30 30 30	61.3±4.03 54.57±2.48 11.2±1.97 8.68±1.98

9. pig japanese b encephalitis-pseudoabies recombinant vaccine ((TK ^-/ gG ^-/ E ⁺)) detect at the intravital immune efficacy of piglet

1) immune programme for children of pig:

Select the healthy negative swinery of 50-60 age in days, be divided into 4 groups, be respectively recombination engineered vaccine immune group (TK of the present invention according to test requirements document ^-/ gG ^-/ E ⁺), vector virus vaccine (TK ^-/ gG ^-/ LacZ ⁺), SA14-14-2 vaccine strain immune group and non-immune group make blank, immunization route is the musculi colli injection, every group of 30 pigs (table 5).Exempt from the back respectively at head and took a blood sample in the 2nd, 4,6,8 weeks through precaval vein, preparation serum, (method is expressed the recombinant pseudorabies virus TK of porcine reproductive and respiratory syndrome virus (PRRSV) GP5 with reference to Fang Liurong etc. to detect the neutralizing antibody of anti-PrV respectively ^-/ gG ^-/ GP5 ⁺Structure and biological characteristics thereof tentatively inquire into. viral journal, 2004,20:249-254) and the humoral immunity level of JEV (method is with reference to Xu etc., Vaccine, 2004,22:1846-1853).Gather anticoagulation simultaneously, separating peripheral blood mononuclear cell (PBMC) detection specificity kills and wounds the BHK-21 cell efficient that infects JEV (method is with reference to Xu etc., Construction of recombinantpseudorabies virus expressing NS1 protein of Japanese encephalitis (SA14-14-2) virusand its safety and immunogenicity.Vaccine, 2004,22:1846-1853), to assess the cellullar immunologic response level that it induces body to produce.

Table 5 pig japanese b encephalitis-pseudoabies recombinant vaccine (TK ^-/ gG ^-/ E ⁺) immune piglet experimental animal echelon design

Group	Immunization protocol
		Vector virus vaccine group (NO.1) JEV SA14-14-2 strain live vaccine group (NO.2) recombinant vaccine immune group of the present invention (NO.3) blank group (NO.4)	With a stature part (10 5.0TCID 50) vector virus vaccine (TK -/ gG -/ LacZ +) immune swine, immunity added with same dosage in back 28 days exempts from once.Carry out immunity according to Chengdu Inst. of Biological Products's product operation instruction, 1 part of every pig immunity, promptly 1mL dosage is with a stature part (10 5.0TCID 50) recombinant vaccine (TK of the present invention preparation -/ gG -/ E +) immune swine, immunity added with same dosage in back 28 days exempts from once.The DMEM substratum of every pig injection and above-mentioned vaccine immunity group equal volume, immune back 28 days with same dosage injection once.

2) PrV neutralizing antibody level detection

Head exempts from back the 4th and the 8th week (two exempt from back the 4th week) precaval vein blood sampling, separation of serum, and (working method is expressed the recombinant pseudorabies virus TK of porcine reproductive and respiratory syndrome virus (PRRSV) GP5 with reference to Fang Liurong etc. to detect the PrV neutralizing antibody ^-/ gG ^-/ GP5 ⁺Structure and biological characteristics thereof tentatively inquire into, viral journal, 2004,20:249-254), the results are shown in Table 6.As can be seen from Table 6, head exempts from 4 weeks of back, and the recombinant vaccine of the present invention preparation can the induction of immunity piglet produces the PrV neutralizing antibody of higher level; Head exempts from 8 weeks of back, and the recombinant vaccine immune group PrV neutralizing antibody level of the present invention's preparation has rising.No matter be that head exempts from 4 weeks of back or head exempts from 8 weeks of back, the recombinant vaccine TK of the present invention's preparation ^-/ gG ^-/ E ⁺Inductive PrV neutralizing antibody and vector virus TK ^-/ gG ^-/ LacZ ⁺Inductive is suitable substantially, and this gG site that shows E gene substitution LacZ gene insertion PrV does not influence TK ^-/ gG ^-Immunogenicity.

Recombinant vaccine (the TK of table 6 the present invention preparation ^-/ gG ^-/ E ⁺) immune piglet PrV neutralizing antibody detected result

Group (30/group)	The neutralizing antibody level
			Head exempts from 4 weeks of back	Head exempts from 8 weeks of back
	TK -/gG -/E + TK -/gG -/LacZ +SA14-14-2 blank group	25.63±4.81 27.54±5.76 0.62±0.25 0.47±0.365			32.76±6.54 35.80±7.41 0.69±0.43 0.52±0.35

3) JEV specific ELISA antibody

Took a blood sample in the 2nd, 4,6 weeks in each immunity back through precaval vein, separation of serum, (method is with reference to Xu etc. to detect ELISA antibody, Construction of recombinant pseudorabies virus expressing NS1 protein of Japaneseencephalitis (SA14-14-2) virus and its safety and immunogenicity, Vaccine, 2004,22:1846-1853), the results are shown in Table 7.As can be seen from Table 7, all can swash JEV specific ELISA antibody behind the recombinant vaccine of the present invention's preparation and the immune weanling pig of encephalitis B attenuated vaccine (SA14-14-2), the antibody horizontal that the recombinant vaccine of the present invention's preparation excites is lower slightly than JEV attenuated live vaccines (SA14-14-2).2 all antibody male rotary rates and antibody horizontal were all lower after head exempted from, head exempts from back 4 all antibody male rotary rates and antibody horizontal all rises to some extent, it is equal 100% that head exempts from recombinant vaccine that back 6 weeks (promptly two exempt from back 2 weeks) the present invention prepares and JEV attenuated live vaccines (SA14-14-2) immune group antibody male rotary rate, and the ELISA antibody titer reaches the highest.

Recombinant vaccine (the TK of table 7 the present invention preparation ^-/ gG ^-/ E ⁺) immune piglet JEV Serum Antibody Detection (ELISA method)

Group (30/group)	Before exempting from	Head exempts from 2 weeks of back				Head exempts from 4 weeks of back				Head exempts from 6 weeks of back
														＜1∶10	1∶10	1∶20	＜1∶20	1∶20	1∶40	1∶80	＜1∶20	1∶20	1∶40	1∶80	1∶160
		TK -/gG -/E + TK -/gG -/LacZ +SA14-14-2 blank group	- a - - -	6/30 b 30/30 6/30 30/30	12/30 0/30 6/30 0/30	12/30 0/30 18/30 0/30	0/30 30/30 0/30 30/30	18/30 0/30 12/30 0/30	6/30 0/30 12/30 0/30	6/30 0/30 6/30 0/30	0/30 30/30 0/30 30/30	0/30 0/30 0/30 0/30	6/30 0/30 6/30 0/30													18/30 0/30 12/30 0/30	6/30 0/30 12/30 0/30

^aWhat represent is that immune piglet is negative at JEV antibody before the immunity; ^bWhat represent is the quantity/total immune quantity that reaches certain antibody titer immunity piglet

4) JEV Specific CTL Cells cytotoxic activity detects

Head exempts from the 8th week of back (two exempt from the 4th week of back), vector virus (TK ^-/ gG ^-/ LacZ ⁺) and blank group CTL activity be respectively 13.307% and 9.21%, and the vaccine strain TK of the present invention preparation ^-/ gG ^-/ E ⁺Be respectively 301.78% and 308.430% with the CTL activity of JEV SA14-14-2 vaccine strain immune group.The recombinant vaccine TK of the present invention's preparation ^-/ gG ^-/ E ⁺With vector virus (TK ^-/ gG ^-/ LacZ ⁺) compare with the blank group, difference is extremely significantly (P＜0.01) all.Above result shows that the recombinant vaccine that the present invention prepares can produce the specific CTL activity of JEV by the induction of immunity piglet, compare with JEV attenuated live vaccines (SA14-14-2) inductive JEV specific CTL activity, low slightly (table 8) of the recombinant vaccine immune group of the present invention's preparation, but difference is not remarkable.

The vaccine TK of table 8 the present invention preparation ^-/ gG ^-/ E ⁺The immunity piglet is brought out JEV Specific CTL Cells cytotoxic activity (X ± SD)

(head exempts from 8 weeks of back)

Group	Number of animals (head)	CTL cytotoxic activity (%)
			JEV SA14-14-2 TK -/gG -/E + TK -/gG -/LacZ +The blank group	30 30 30 30	308.430±6.42 301.78±4.61 13.307±1.66 9.21±3.330

In a word, the test-results of specific embodiment shows among the present invention, pig japanese b encephalitis of the present invention-pseudoabies recombinant vaccine energy excitating organism produces the neutralizing antibody of the high Pseudorabies virus of tiring, and produce, and there is not significant difference with immune efficacy that JEV attenuated live vaccines strain SA14-14-2 immune group produces at pig japanese b encephalitis virus-specific ELISA antibody and specific CTL activity.

Claims (4)

1, a kind of recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain Pseudorabies virus WKQ-JE-E001, its deposit number is CCTCC-V200603.

2, a kind of recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain according to claim 1, it is characterized in that, the main virulence gene TK of described recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strain disappearance Pseudorabies virus does not produce functional glycoprotein gG.

3, the vaccine that comprises claim 1 or 2 described recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strains.

4, claim 1 or the 2 described a kind of recombinant pig japanese b encephalitis-aujeszkys disease geneticly engineered strains application in preparation pig japanese b encephalitis-pseudoabies recombinant vaccine.

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