CN109439634A - Pseudorabies virus genetic engineering attenuated vaccine strain and its application - Google Patents

Pseudorabies virus genetic engineering attenuated vaccine strain and its application Download PDF

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CN109439634A
CN109439634A CN201811330929.5A CN201811330929A CN109439634A CN 109439634 A CN109439634 A CN 109439634A CN 201811330929 A CN201811330929 A CN 201811330929A CN 109439634 A CN109439634 A CN 109439634A
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pseudorabies virus
gene
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virus
ah02la
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CN109439634B (en
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王继春
刘娅梅
张传健
陈赛赛
乔永峰
呙明鹏
王志胜
许梦微
郑亚婷
郭容利
侯继波
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Jiangsu Academy of Agricultural Sciences
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Abstract

The present invention provides pseudorabies virus genetic engineering attenuated vaccine strain and its application, belongs to animal medicine vaccines arts.The vaccine strain, be by the gC gene replacement in pseudorabies virus Bartha K61 pnca gene group be AH02LA plants of pseudorabies virus gC gene, obtain after the gD gene that gD gene replacement is AH02LA plants of pseudorabies virus, described pseudorabies virus AH02LA plants of gC gene order is as shown in SEQ ID No:1, and described pseudorabies virus AH02LA plants of gD gene order is as shown in SEQ ID No:2.Vaccine strain of the present invention can be good at adapting to ST cell, and titre is high, and safety is higher；100% protection can be provided to immune pig after using the vaccine strain as the vaccine immunity of effective component, which can not only prevent to fall ill, moreover it is possible to significantly reduce toxin expelling rate, shedding virus is greatly decreased, toxin expelling time greatly shortens.

Description

Pseudorabies virus genetic engineering attenuated vaccine strain and its application
Technical field
The invention belongs to the vaccines arts of animal medicine, and in particular to pseudorabies virus genetic engineering attenuated vaccine strain and It is applied.
Background technique
Pseudoabies is that one kind caused by pseudorabies virus (Pseudorabies Virus, PRV) is acute, strong biography It catches an illness.It is typical that the domestic animals such as ox, sheep and dog, cat and wild animal occur that pseudoabies shows as nervous symptoms often and surprise is itched etc. Symptom, with clinical symptoms such as fever and encephalomyelitis.Pig is the unique storage host of pseudorabies virus, porcine pseudorabies Symptom is age-related, and newborn piglet has nervous symptoms, and the death rate is high；Growing and fattening pigs are mainly respiratory symptom, Adult Pig infection Death does not occur usually for growth retardation after Pseudorabies virus, and sow is often miscarried, and orchitis occurs for boar, and virus can lead to Cross sperm propagation.PRV gene delection strain vaccine (K61 plants of the Bartha of such as gE gene delection) cooperation identifies immune swine and open country The ELISA detection method of the PRV gE antibody of poison infection pig can easily be diagnosed to be whether have wild virus infection, can be by continuing It eliminates positive boar and establishes sweeping field, by effort in more than 10 years, many countries realized pseudo- mad in domestic pig group in the world The purification of dog disease virus, after China, 2000, many pig farms have also gradually been successfully established the pig of PRV gE negative antibody Group, many pig farms realize the purification of PRV substantially, and porcine pseudorabies have obtained preferable control.
Since 2011, there is porcine pseudorabies epidemic situation on a large scale in many pig farms of northern China, later epidemic situation rapidly to The sprawling of national major part pig farm, although pig farm takes the measures such as reinforced immunological, PRV gE antibody positive rate is high, China's pig breeding industry is caused to seriously endanger.The result of study of multiple team proves that this wheel epidemic situation is drawn by the PRV street strain to make a variation It rises, the variant is pathogenic to be significantly increased, although K61 plants of traditional vaccine strain Bartha can provide clinical protection, cannot be prevented The toxin expelling of immunity inoculation animal.
Summary of the invention
The object of the present invention is to provide pseudorabies virus genetic engineering attenuated vaccine strain, which can be good at adapting to ST cell, titre is high, and since the attenuated vaccine strain is the transformation carried out to K61 plants of Bartha, safety is higher.
It is a further object of the present invention to provide contain pseudorabies virus genetic engineering attenuated vaccine strain pseudorabies vaccines, Using that can provide immune pig 100% protection after the vaccine immunity, which can not only prevent to fall ill, moreover it is possible to significantly reduce Shedding virus is greatly decreased in toxin expelling rate, and toxin expelling time greatly shortens, and since the strain applies classics vaccine strain Bartha K61 Strain is skeleton, so the safety of the recombination low virulent strain is good.
The purpose of the present invention adopts the following technical scheme that realization.
Pseudorabies virus genetic engineering attenuated vaccine strain, being will be in pseudorabies virus Bartha K61 pnca gene group GC gene that gC gene replacement is AH02LA plants of pseudorabies virus, the gD that gD gene replacement is AH02LA plants of pseudorabies virus It is obtained after gene, described pseudorabies virus AH02LA plants of gC gene order is as shown in SEQ ID No:1, the mad dog of puppet The gD gene order of sick AH02LA plants of virus is as shown in SEQ ID No:2.
The present invention also provides the pseudorabies virus genetic engineering attenuated vaccine strains in preparing pseudorabies vaccines Using.
The present invention also provides using the pseudorabies virus genetic engineering attenuated vaccine strain as the vaccine of active constituent.
In the present invention, pseudorabies virus genetic engineering attenuated vaccine strain virus liquid is prepared with the following method: will be pseudo- Rabies virus gene engineering attenuated vaccine strain is inoculated with ST cell, using the DMEM culture medium culture 36~48 containing fetal calf serum Hour, viral cultures are harvested, centrifuging and taking supernatant is as pseudorabies virus genetic engineering attenuated vaccine strain virus after freeze thawing Liquid.
In the present invention, the vaccine is live vaccine.Immunologic adjuvant and vaccine excipients etc., ability can be added in vaccine Field technique personnel can be easy to carry out production.
The present invention also provides a kind of recombinant vector live vaccine, active constituent is in the pseudorabies virus genetic engineering It inserts in attenuated vaccine strain genome and obtains after external source protective antigen gene expression cassette.Promoter can choose pMCMV IE, SV40 and pHCMV IE etc., the insertion point of foreign gene includes between the UL22 of genome and UL21, UL46 and UL27 it Between, between UL51 and UL50, between UL35 and UL36, between UL40 and UL41, between UL44 and UL26.5 and UL4 and UL3.5 Between noncoding region, the foreign gene of insertion include the E protein gene of swine fever virus, pig circular ring virus Cap protein gene, The G-protein gene of hydrophobin, the VP1 gene of foot and mouth disease virus, the HA gene of swine influenza virus and the VP60 of Rabbit pest virus Gene etc..
Applicant passes through a large amount of creative labour, obtains pseudorabies virus gD and gC genetic recombination low virulent strain (B-gD&gCSStrain).B-gD&gCSStrain be with K61 plant of Bartha for skeleton, gB, gG, gH, gK, gL, gM and gN gene and Bartha K61 completely the same (GenBank ID:JF797217.1)；Itself gC and gD gene is to take gene engineering method that will become Different strain gene replacement and obtain, with AH02LA plants it is completely the same (gC gene order be SEQ ID No:1；GD gene order is SEQ ID No:2), with variation strain's very high homology is now disclosed.B-gD&gC of the present inventionSStrain is building PRV Bartha K61 first The bacterial artificial chromosome of strain, the gD gene for being then variant AH02LA plants by gD gene replacement, finally again by its gC gene Replace with AH02LA plants of variant of gC gene, at the same Revive virus and obtain.Pseudorabies virus B-gD&gCSStrain can be fine Ground adapts to ST cell, grows titre up to 108.0TCID50/ mL or more.Pseudorabies virus B-gD&gCSThe live vaccination of strain is disconnected Efficient protection, no body temperature reaction can be quickly generated after milk PRV feminine gender piglet, no clinical symptoms make a variation to pseudorabies virus Strain 100% is protected, and especially the vaccine can not only prevent to fall ill, moreover it is possible to prevent toxin expelling or shedding virus from being greatly decreased, toxin expelling time It greatly shortens, achieves unexpected technical effect.It is inoculated with pseudorabies virus B-gD&gCSIt carries out attacking poison protection examination after strain Test the result shows that, animal and the infection animal of the vaccine can be immunized by being monitored to distinguish to PRV gE and gB antibody, it is non- Often be conducive to the purification to PRV variant.It is used as the protective antigens of the live vector expression susceptible cause of disease of pig, moreover it is possible to reach and connect One needle of kind prevents the effect of two or more epidemic diseases.Due to B-gD&gCSThe skeleton of strain is widely used, highly-safe K61 plants of Bartha, therefore be the reliable vaccine candidate low virulent strain of one plant of safety.
Detailed description of the invention
Fig. 1, pseudorabies virus B-gD&gCSStrain building schematic diagram.(A) Mini-F is inserted by PRV by homologous recombination The loci of K61 plants of gC genes of Bartha.(B) it is recombinated by the first round by the AH02LA strain containing kalamycin resistance gene Bartha plants of gD gene replacement of gD gene, then the second wheel recombination knocks out kalamycin resistance gene, constructs successfully gD and replaces Change strain clone (BAC pB-gDS-mini-F).(C) again by homologous recombination by BAC pB-gDSMini-F sequence in-mini-F Column replace with AH02LA plants of gC gene, are built into bis- replacement strain (the i.e. PRV B-gD&gC of gC and gDSStrain).
Fig. 2, transfer vector pHA2-Puc19-H1B-H2BStructure chart, EGFP be fluorescent protein expression box.
B-mini-F plants of electromicroscopic photograph under Fig. 3,488nm ultraviolet excitation, as shown in the figure is to issue in ultraviolet excitation The mini-F of fluorescence recombinates K61 plants of PRV Bartha (i.e. PRV B-mini-F plants) virus plaques out.
The RFLP of Fig. 4, PRV Bartha K61BAC and its recon scheme.Left side is illustrated as pB-mini-F plants of (1 Hes of sering 4)、BAC pB-gDS- KAN-mini-F plants (serings 2 and 5) and BAC pB-gDSThe RFLP figure of-mini-F plants of (sering 3 and 6) DNA Spectrum, respectively through Hind III (sering 1-3) digestion or Sph I digestion (sering 4-6), arrow show the item that size variation occurs Band.Right figure show the prognostic chart of Reference Strains (K61 plants of Bartha) sequence (GenBank ID:JF797217.1) rflp analysis Spectrum, Mark stripe size is 1K, 2K, 5K, 10K and 100M from bottom to top.
Fig. 5, pseudorabies virus gD and gC genetic recombination low virulent strain (B-gD&gCSStrain) PCR qualification figure.Left figure is shown K61 plants of Bartha and PRV B-gD&gCSThe PCR of strain gD gene identifies electrophoretogram (the primer AH02LA-gD-F/ AH02LA-gD-R).Right figure show K61 plants of Bartha and PRV B-gD&gCSThe PCR identification electrophoretogram of strain gC gene is (used Primer is SEQ-AH02LA gC F/SEQ-AH02LA gC R).
Fig. 6, pseudorabies virus gD and gC genetic recombination low virulent strain (B-gD&gCSStrain) virus plaques.Institute in upper figure circle It is shown as PRV B-gDS- mini-F strain virus plaque (under ultraviolet excitation), the following figure square shown in be PRV B-gD&gCSDisease Malicious plaque (under white light).
Fig. 7, PRV B-gD&gCSThe growth characteristics of cultured of strain and parental virus (K61 plants of PRV Bartha) compare.
Specific 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 building of K61 plants of pseudorabies virus Bartha of bacterial artificial chromosome
1, the acquisition of transfer vector
For mini-F carrier is inserted into K61 plants of pseudorabies virus PRV Bartha of gC allele site (Fig. 1), It designs gC gene upstream and downstream homology arm primer (Bartha-gC-H1-F/R and Bartha-gC-H2-F/R) (table 1), passes through PCR Sequence is obtained, then by digestion and is connected on pUC19 carrier, then reconnects mini-F carrier (Fig. 2), transfer is obtained and carries Body.
1 PCR of table and sequencing primer
First using the DNA of pseudorabies virus Bartha K61 plants (inspection nose swab sample in pig farm is isolated) as mould The upstream of plate, PCR amplification gC gene (the 54635th nucleotide sequence of 53193- in Bartha K61 pnca gene group) is homologous Arm pieces section H1, PCR reaction system (table 2) and response procedures are as follows.
2 25 μ l PCR reaction system of table
Sample Volume
LA Taq DNA polymerase 0.25μl
2×GC Buffer II 12.5μl
dNTPs(2.5mM) 4μl
Upstream primer Bartha-gC-H1-F 1μl
Downstream primer Bartha-gC-H1-R 1μl
Template DNA 1μl
ddH2O 5.25μl
LA Taq archaeal dna polymerase, 2 × GC Buffer II in PCR reaction system are purchased from Takara company, primer sequence Column are shown in Table 1.Reaction system is mixed, PCR amplification is carried out, response procedures are as follows: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 40s, 57 DEG C annealing 40s, 72 DEG C of extensions 1min30s, 30 recycle；72 DEG C of extension 10min.PCR product is subjected to 1% Ago-Gel electricity Swimming, discovery is there are the purpose band of 1200bp or so and is cut, and is recycled using DNA gel QIAquick Gel Extraction Kit, after digestion It is connected on pUC19 carrier, obtains pUC19-H1.
Then, using K61 plants of pseudorabies virus Bartha of DNA as template, using primer Bartha-gC-H2-F and Bartha-gC-H2-R expands the gC gene (53193- the 54635th in Bartha K61 pnca gene group by above-mentioned same procedure Position nucleotide sequence) downstream homology arm segment H2, and downstream homology arm segment H2 is connected on pUC19-H1, obtain pUC19- H1-H2。
PUC19-H1-H2 Plasmid DNA and pHA2 Plasmid DNA (are disclosed in Wang et al；Virus Research 159 (2011)23-31,Generation of an infectious clone of duck enteritis virus(DEV)and Of a vectored DEV expressing hemagglutinin of H5N1avian influenza virus) respectively Using Pac I digestion, metastasis transplanting physique grain pHA2-Puc19-H1 is obtained after connectionB-H2B.As shown in Fig. 2, metastasis transplanting physique grain pHA2-Puc19-H1B-H2BIn upstream and downstream homology arm between have fluorescent protein expression box, transfer vector sequence meets expection.
2, K61 plants of pHA2 recombinant pseudorabies virus Bartha of acquisition
Primary chicken embryo fibroblasts (Chicken embryo is prepared using 9~10 age in days SPF chicken embryos Fibroblasts, CEF), after cell grows up to single layer, transfected.It is transfected according to conventional calcium phosphate infection protocol, by 0.5~ 1 μ g metastasis transplanting physique grain pHA2-Puc19-H1B-H2BThe DNA of K61 plants of pseudorabies virus Bartha of DNA and 0.2~1 μ g are total Transfected primary CEF carries out homologous recombination, and culture solution is absorbed after 24 hours, is added and contains 10% fetal calf serum and 0.5% Methyl cellulose The DMEM culture medium covering of element, continues 24~48h of culture, and picking issues green fluorescence in the case where wavelength is 488nm ultraviolet excitation Recombinant virus plaque (Fig. 3), be inoculated into fresh CEF, so repeat 3 circulation more than, obtain the recombinant virus of purifying, order Entitled PRV B-mini-F plants.
Therefore, PRV B-mini-F plants be by K61 plants of pseudorabies virus PRV Bartha of gC gene replacement at It is obtained after mini-F carrier.
3, the acquisition and identification of pseudorabies virus Bartha K61 plants of bacterial artificial chromosome
The DNA for taking 1~3 B-mini-F plants of μ g recombinant virus is transferred to competent cell E.coli DH10B by electrotransformation (being purchased from Invitrogen company), extracts the DNA of positive colony, carries out RFLP points after Hind III and Sph I distinguish digestion Analysis shows to coincide with prediction map, then extracts DNA, is transferred in competent cell E.coli GS1783 again by electrotransformation, Take one of clone, extract DNA, carried out after Hind III and Sph I distinguish digestion using same method rflp analysis (see Fig. 4), it as a result coincide with prediction map, is pB-mini-F plants by the clone designation.
The building and identification of embodiment two, pseudorabies virus genetic engineering attenuated vaccine strain
1, the clone of PRV AH02LA plants of gD genes and identification
For the gD gene for being AH02LA plants of pseudorabies virus variant by the gD gene replacement in recombinant bacterium pB-mini-F Sequence, using AH02LA plants of pseudorabies virus variant (deposit number is CGMCC No.10891) DNA as template, with AH02LA-gD-F and AH02LA-gD-R be AH02LA plants of primer pair pseudorabies virus variant gD gene (SEQ ID No: 2) PCR amplification is carried out, PCR reaction system (table 3) and response procedures are as follows.
3 25 μ l PCR reaction system of table
Sample Volume
LA Taq DNA polymerase 0.25μl
2×GC Buffer II 12.5μl
dNTPs(2.5mM) 4μl
Upstream primer AH02LA-gD-F 1μl
Downstream primer AH02LA-gD-R 1μl
Template DNA 1μl
ddH2O 5.25μl
LA Taq DNA polymerase, 2 × GC Buffer II in PCR reaction system are purchased from Takara company, primer sequence Column are shown in Table 1.Reaction system is mixed, PCR amplification is carried out, response procedures are as follows: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 40s, 57 DEG C annealing 40s, 72 DEG C of extensions 2min10s, 30 recycle；72 DEG C of extension 10min.PCR product is subjected to 1% Ago-Gel electricity Purpose band is connected to carrier T pMD19 and (on (purchased from Takara), obtains pMD19-gD by swimmingA, through Nanjing Jin Sirui biology section Skill Co., Ltd sequence verification.
In order to be AH02LA plants of pseudorabies virus variant of gD gene by the gD gene replacement in pB-mini-F plants, Using pair of primers Kan ins gD F and Kan ins gD R (table 1), with pDEVC-KCE-H5(UL55)KANDNA is that template is (public It opens in Wang et al；Virology Journal (2015) 12:126, Construction of a recombinant duck enteritisvirus(DEV)expressing hemagglutinin of H5N1avian influenza virus based on an infectious cloneof DEV vaccine strain and evaluation of its Efficacy in ducks and chickens), card of the PCR amplification one end with Partial Fragment c in AH02LA plants of gD genes That mycin resistant gene (is abbreviated as c-sm segment, i.e. part in Fig. 1 between (B) first sequence two *, wherein sm is card The abbreviation of that mycin resistant gene), PCR reaction system (table 4) and response procedures are as follows.
4 25 μ l PCR reaction system of table
Sample Volume
LA Taq DNA 0.25μl
2×GC Buffer II 12.5μl
dNTPs(2.5mM) 4μl
Upstream primer Kan ins gD F 1μl
Downstream primer Kan ins gD R 1μl
Template DNA 1μl
ddH2O 5.25μl
LA Taq DNA polymerase, 2 × GC Buffer II in PCR reaction system are purchased from Takara company, primer sequence Column are shown in Table 1.Reaction system is mixed, PCR amplification is carried out, response procedures are as follows: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 40s, 57 DEG C annealing 40s, 72 DEG C of extensions 2min10s, 30 recycle；72 DEG C of extension 10min.PCR product is subjected to 1% Ago-Gel electricity Swimming, recycling purpose band (c-sm segment) are connected to the pMD19-gD also through Sac I digestion after Sac I digestionAIn segment, i.e., C-sm segment is inserted into AH02LA plants of gD gene internals and two terminal sequence of sm segment is segment c.The positive colony of acquisition is passed through Nanjing Genscript Biotechnology Co., Ltd.'s sequence verification, is named as pMD19-gDA-KAN。
2, gD recombinates the acquisition and identification of the BAC recon of pB-mini-F
First with pMD19-gDAThe DNA of-KAN is template, using pair of primers Bartha ins gDA- kan F and Bartha ins gDA- kan R carries out PCR, the internal AH02LA strain gD gene piece for inserting kalamycin resistance gene of amplification Section, the segment both ends are respectively provided with K61 plants of gD genes of Bartha (119436- the 120638th in Bartha K61 pnca gene group Position nucleotide sequence) upstream and downstream 40bp homologous sequence (see first bar segment of part (B) in Fig. 1).
The competent cell that electricity is transferred to the GS1783 containing pB-mini-F after PCR fragment is recovered and Dpn I digestion carries out the K61 plants of gD gene replacements of Bartha, are inserted the AH02LA strain gD of kalamycin resistance gene by one wheel homologous recombination inside Genetic fragment (Fig. 1), the positive colony name of acquisition are as follows: BAC pB-gDS-KAN-mini-F.Then inserting card inside, that is mould The second wheel recombination is carried out inside the AH02LA strain gD genetic fragment of plain resistant gene, since sm segment both ends are pieces in the segment Section c, therefore when second of homologous recombination, a c segment and kalamycin resistance gene are knocked out, screening has chloramphenicol anti- Property, and there is no the positive monoclonal of resistance to kalamycin, it is named as BAC pB-gDS-mini-F.The positive monoclonal, warp RFLP atlas analysis is carried out after Hind III and Sph I digestion, with expected mutually attached (Fig. 4).Replaced gD gene is through PCR and survey Sequence verifying, it is consistent with AH02LA plants.
3, the acquisition and identification of pseudorabies virus gD and gC recombinant strain
First using AH02LA plants of PRV of DNA as template, using primer Bartha-gC-H1-F and Bartha-gC-H2-R PCR amplification is carried out, AH02LA plants of gC genetic fragments (SEQ ID NO:1), two terminal sequences and BAC pB-gD are obtainedS-mini-F Middle two terminal sequence of mini-F has homology, by the amplified fragments of recycling and BAC pB-gDSThe DNA cotransfection ST of-mini-F is thin Born of the same parents, while Revive virus, by BAC pB-gDSMini-F in-mini-F replaces with AH02LA plants of variant of gC gene ((C) in Fig. 1).It is observed under ultraviolet light (488nm) excitation within 1~2 day after transfection, observes the virus plaques for issuing green fluorescence For the virus that gC is not replaced, chooses spot purifying and obtain a strain virus, be named as PRV B-gDS- mini-F plants.Observe do not issue it is glimmering The virus plaques (Fig. 6) of light choose spot through 3 wheels, obtain the virus of one plant of purifying, be named as PRV B-gD&gCSStrain.B-gD&gCSStrain GD and gC gene through PCR and sequence verification (Fig. 5), it is consistent with AH02LA plants of variant.
In conclusion being pseudorabies virus by the gC gene replacement in pseudorabies virus Bartha K61 pnca gene group AH02LA plants of gC gene (SEQ ID NO:1), gD gene (the SEQ ID that gD gene replacement is pseudorabies virus AH02LA plants NO:2 after), PRV B-gD&gC has been obtainedSStrain.
Embodiment three, PRV B-gD&gCSThe growth characteristics identification of strain
By PRV B-gD&gCSK61 plants of strain and PRV Bartha cover with the ST cell of single layer with MOI respectively for 0.01 inoculation On, set 5%CO2In constant incubator after 37 DEG C of incubation 1h, supernatant is sucked, after washing 3 times using PBS, changes cell maintenance into The liquid DMEM culture medium (Gibco) of (contain 3% fetal calf serum (FBS)) is cultivated, and after inoculation 0h, 6h, 12h, For 24 hours, 36h, 48h and 72h collect culture supernatant and cell mixture, after -70 DEG C and 37 DEG C of freeze thawing, 5000rpm centrifugation 10min takes supernatant, and inoculation grows up to the fresh ST cell of single layer, detects the TCID of virus50, it is repeated 3 times altogether.Make the growth of virus Curve.
It can be seen that PRV B-gD&gC from the growth curve (Fig. 7) of virusSStrain has phase with K61 plants of PRV Bartha As growth kinetics, the two compares no significant difference.36h~48h reaches peak value after inoculation, and titre all reaches 108.0TCID50/ mL or more is able to satisfy the requirement of live vaccine production.
Example IV, PRV B-gD&gCSThe safety of strain and immune efficacy
1, test material
1.1 viruses and pseudorabies living vaccines
PRV B-gD&gCSStrain virus liquid (with embodiment three), viral level 108.25TCID50/mL.Preparation method is such as Under: by PRV B-gD&gCSStrain inoculation ST cell, using the culture of DMEM culture medium 36~48 hours containing 3% fetal calf serum, Viral cultures are harvested, centrifuging and taking supernatant is after freeze thawing up to virus liquid.
Pseudorabies living vaccines (K61 plants of Bartha), viral level 108.50TCID50/mL。
AH02LA plants of PRV, it is used for challenge test, viral level 107.00TCID50/mL。
1.2 diagnostic kit
PRV gE antibody diagnosing reagent kit, PRV gB antibody diagnosing reagent kit, pig breeding and respiratory disorder syndrome virus (PRRSV) antibody diagnosing reagent kit and swine fever virus (SFV) antibody diagnosing reagent kit are purchased from the prosperous biological section of Beijing Ai Deshi member Skill Co., Ltd (IDEXX) company.
2, experimental animal
28~35 age in days sodium selenites 20, PRV antigen and PRV gE and gB antibody are feminine gender, pig breeding and breathing Distress syndrome virus (PRRSV) and swine fever virus (SFV) antigen and antibody are feminine gender.The preceding diet for verifying pig of inoculation, Mental status and clinical setting etc., all unsound pigs exclude test.It is marked by ear tag number and cage number with circle number Know.The sub-cage rearing in isolation environment.Feed is perfect compound feed (commercial product), is drunk water for tap water, the daily morning and It respectively feeds afternoon once.Special messenger is responsible for raising, carries out 2 sanitation and hygiene daily.
3, it is grouped
Experimental animal once introduces, and is randomly divided into A~D group, every group 5, is specifically shown in Table 5.
4, the index of test method and research
(1) test method
It is immune: by PRV B-gD&gCSThe sterile DMEM of application is trained respectively for strain virus liquid and PRV Bartha K61 strain virus liquid Nutrient solution is diluted to 106.00TCID50For/2mL for being immunized, immunization ways are intramuscular injection, and immunizing dose is 2mL/ head.A group connects Kind PRV B-gD&gCSStrain, B group are inoculated with K61 plants of PRV Bartha, and C group (attacking malicious control group) is inoculated with 2mL DMEM liquid, and D group is not Inoculation is negative control group (table 5).
Attack poison: all pigs in addition to negative control group attack poison in 1 week after vaccine inoculation, using AH02LA plants of PRV, with 2LD50/ head collunarium attacks poison, and every 2mL is observed 14 days after attacking poison.
Serum gB and gE antibody test: it before immune, attack before poison and (is attacked after poison 14 days) at the end of attacking poison three time points All pig blood serum samples are taken, detect PRV gE and gB antibody using kit.
Clinicing symptom observation: all test pigs observe spirit, diet and various respiratory systems, nervous system daily With systemic clinical manifestation.
Measurement of bldy temperature: since before vaccine inoculation, until off-test (attacking after poison 14 days), all pigs measure body temperature daily Once (rectal temperature).
The detection of toxin expelling situation: it before attacking poison, attacks after poison to off-test (attack poison after 14 days), acquires pig nose swab sample daily Product, sample set -70 DEG C of preservations, when detection after doubling dilution inoculation BHK-21 cell observation lesion to determine whether discharge virus, And the titre of toxin expelling.
(2) index studied
Disease incidence, the death rate, body temperature rising condition, toxin expelling situation, serum antibody sun turn situation, pulmonary lesion.
5, test result
PRV gB and the gE antibody of all test pigs is feminine gender, negative control group (D group) pig to examination before vaccine inoculation PRV gB and gE antibody at the end of testing is feminine gender, after vaccine inoculation at 1 week (before attacking poison) A group and B group all tests The PRV gB antibody of pig is the positive, and attacking after poison all immune group pigs and attacking the PRV gE antibody of malicious control group survival pig is sun Property (table 5).
After attacking poison, negative control group (D group) pig is dead without morbidity, and no body temperature increases, and toxin expelling (data omission) is not detected； It attacks 5 pigs of malicious control group all to occur subtracting food the 2nd day after attacking poison, spirit is depressed, sneezing, has a running nose and has difficulty in breathing Etc. symptoms, body temperature be all increased to 40.5 DEG C or more, detect toxin expelling from nose swab sample, shedding virus is up to 101.30~108.83/ Gram, each dead 1 on the the 5th, 6 and 7 after attacking poison, dead pig and survival pig (attack after poison 14) dissect Jun Jianyou lung typical case and go out Blood lesion shows that experimental control is set up.After attacking poison, A group and B group test pig are without clinical symptoms, and no body temperature increases, A group, B group Typical cytopathic is had no with D group test pig dissect lung, and all test pig dissects of C group lung sees there is typical bleeding symptom (table 5).Toxin expelling all occurs after attacking poison for B group pig, and toxin expelling titre is up to 109.82TCID50/ gram, the toxin expelling duration is for up to 10 It；And A group pig has 2 toxin expelling does not occur after attacking poison, the raw toxin expelling of another 3 hair, toxin expelling titre is up to 106.19TCID50/ gram, It is remarkably decreased compared with B group, toxin expelling time is no more than 7 days, is obviously shortened (table 6) compared with B group.It is above-mentioned the experimental results showed that PRV B-gD& gCSStrain protects the metainfective toxin expelling of variant and is significantly increased than Bartha K61 plants, is very beneficial for the prevention and control of variant And purification.
5 safety of table and immuning effect test result
Note: before B.V. refers to vaccine inoculation in table 5；P.V. after referring to vaccine inoculation；P.C. refer to after attacking poison；GB+ refers to that PRV gB is anti- Body is positive；GE+ refers to PRV gE antibody positive；"/" refers to not applicable；" a " refers to positive pig quantity；" b " refers to test pig sum in group.
Table 6 attacks A, B, C group toxin expelling result after poison
Note: "/" indicates not applicable in table 6；Toxin expelling is not detected in "-" expression；Virus titer is TCID50/ gram.
In conclusion pseudorabies virus genetic engineering low virulent strain (B-gD&gCSStrain) malicious variant wild to PRV can be generated AH02LA plants of complete protection, no body temperature reaction, no clinical symptoms, toxin expelling rate significantly reduces and shedding virus is greatly decreased, toxin expelling Time greatly shortens, and K61 plants of PRV Bartha can only generating unit code insurance shield, toxin expelling cannot be prevented, B-gD&gCSStrain is used as epidemic disease Seedling strain is significantly better than K61 plants of Bartha to the protecting effect of variant.PRV B-gD&gCSThe live vaccination pig of strain preparation PRV gB antibody is detected, PRV gE antibody is not detected, is a kind of DIVA that can identify vaccine immunity animal Yu wild virus infection animal Vaccine.Due to B-gD&gCSThe skeleton of strain is widely used Bartha K61 plants, and safety is reliable, therefore is one plant ideal Vaccine candidate low virulent strain, promoting and applying will be very beneficial for carrying out the purification of PRV variant in pig farm, have a extensive future.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>pseudorabies virus genetic engineering attenuated vaccine strain and its application
<130> 201811091
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1464
<212> DNA
<213>pseudorabies virus AH02LA plants
<400> 1
atggcctcgc tcgcgcgtgc gatgctcgcg ctgctggcgc tctacacggc ggccatcgcc 60
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agcagcgcgg gcgagctctc gccctcgccg ccctcgacgc ccgagcccgt ctcggggacg 180
acgggggccg cggcctccac gcccgccgcc gtctcgacgc cccgggtccc gccgccctcg 240
gtctcgcgcc ggaagcccca gcggaacggc aacaggacgc gcgtccacgg cgacaaggcc 300
acctcgcacg ggcgcaagcg catcgtgtgc cgcgagcggc tgttctcggc gagggtgggg 360
gacgcggtca gcttcgggtg cgccgtcgtc ccgcgcgccg gggagacctt cgaggtccgc 420
ttctgccgcc gcgggcgctt ccgctcgccc gacgccgacc ccgagtactt tgacgagccc 480
ccgcgcccgg agctcccgcg ggagcggctc ctcttcagct ccgccaacgc ctccctcgcc 540
cacgcggacg cgctcgcctc cgccgtcgtc gtcgagggcg agcgcgcgac cgtcgccaac 600
gtctcgggcg aggtgtccgt gcgcgtggcc gcggcggacg ccgagaccga gggcgtctac 660
acgtggcgcg tgctgtccgc caacggcacc gaggtccgca gcgccaacgt ctcgctcgtc 720
ctgtaccacc agcccgagtt cggcctgagc gcgccgcccg tcctcttcgg cgagcccttc 780
cgggcggtgt gcgtcgtccg cgactactac ccgcggcgca gcgtgcgcct gcgctggttc 840
gcggacgagc acccggtgga cgccgccttc gtgaccaaca gcaccgtggc cgacgagctc 900
gggcgccgca cgcgcgtctc cgtggtgaac gtgacgcgcg cggacgtccc gggcctcgcg 960
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gccgagcacc ccggcctgct caacgtgcgg agcgcccgcc cgctgtcgga cctcgacggg 1260
cccgtcgact acacctgccg cctcgagggc atgccctcgc agctgcccat cttcgaggac 1320
acgcagcgct acgacgcctc ccccacgtcc gtgagctggc ccgtcgtgac cagcatgatc 1380
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ctgacgctga cgacggtccc ctcgcccttc gtcggccccg cggacgtcta ccacacgcgc 180
ccgctggagg acccgtgcgg ggtggtggcg ctgatctccg acccgcaggt ggaccggctg 240
ctgaacgagg cggtggccca ccggcggccc acgtaccgcg cccacgtggc ctggtaccgc 300
atcgcggacg ggtgcgcgca cctgctgtac tttatcgagt acgccgactg cgaccccagg 360
cagatctttg ggcgctgccg gcgccgcacc acgccgatgt ggtggacccc gtccgcggac 420
tacatgttcc ccacggagga cgagctgggg ctgctcatgg tggccccggg gcggttcaac 480
gagggccagt accggcgcct ggtgtccgtc gacggcgtga acatcctcac cgacttcatg 540
gtggcgctcc ccgaggggca agagtgcccg ttcgcccgcg tggaccagca ccgcacgtac 600
aagttcggcg cgtgctggag cgacgacagc ttcaagcggg gcgtggacgt gatgcgattc 660
ctgacgccgt tctaccagca gcccccgcac cgggaggtgg tgaactactg gtaccgcaag 720
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cggccctcgg cgggctcgcc gaggcccagg ccccggcccc ggcccaggcc ccggccgaag 840
cccgagcccg ccccggcgac gcccgcgccc cccggccgcc tgcccgagcc ggcgacgcgg 900
gaccacgccg ccggggggcg ccccacgccg cgacccccga ggcccgagac gccgcaccgc 960
cccttcgccc cgccggccgt cgtgcccagc gggtggccgc agcccgcgga gccgttcccg 1020
ccccggacca ccgccgcgcc gggcgtctcg cgccaccgct cggtgatcgt cggcacgggc 1080
accgcgatgg gcgcgctcct ggtgggcgtg tgcgtctaca tcttcttccg cctgaggggg 1140
gcgaaggggt atcgcctcct gggcggtccc gcggacgccg acgagctaaa agcgcagccc 1200
ggtccgtag 1209

Claims (6)

1. pseudorabies virus genetic engineering attenuated vaccine strain is by the gC in pseudorabies virus Bartha K61 pnca gene group GC gene that gene replacement is AH02LA plants of pseudorabies virus, the gD base that gD gene replacement is AH02LA plants of pseudorabies virus It is obtained because after, described pseudorabies virus AH02LA plants of gC gene order is as shown in SEQ ID No:1, the pseudoabies Viral AH02LA plants of gD gene order is as shown in SEQ ID No:2.
2. pseudorabies virus genetic engineering attenuated vaccine strain described in claim 1 is preparing the application in pseudorabies vaccines.
3. using pseudorabies virus genetic engineering attenuated vaccine strain described in claim 1 as the vaccine of active constituent.
4. vaccine according to claim 3, it is characterised in that pseudorabies virus genetic engineering attenuated vaccine strain virus liquid is adopted It prepares with the following method: pseudorabies virus genetic engineering attenuated vaccine strain being inoculated with ST cell, using containing fetal calf serum DMEM culture medium culture 36~48 hours harvests viral cultures, and centrifuging and taking supernatant is as pseudorabies virus base after freeze thawing Because of engineering attenuated vaccine strain 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 constituent is the pseudorabies virus base described in claim 1 It is obtained after external source protective antigen gene expression cassette because being inserted in engineering attenuated vaccine strain genome.
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CN110029079A (en) * 2019-03-27 2019-07-19 南京农业大学 The recombined bacillus subtilis in expression secretion porcine pseudorabies toxalbumin dominant antigen area and application
CN110387354A (en) * 2019-08-16 2019-10-29 江苏省农业科学院 Pseudorabies virus Attenuation strain and its application
CN111154917A (en) * 2020-02-22 2020-05-15 南京农业大学 PCR primer and method for identifying porcine pseudorabies virus variant strain
CN111690619A (en) * 2020-04-20 2020-09-22 华南农业大学 Recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes and construction and application thereof
CN112430625A (en) * 2020-11-23 2021-03-02 武汉枢密脑科学技术有限公司 Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gD protein gene, virus, preparation method and application thereof
CN112481223A (en) * 2020-12-01 2021-03-12 江苏省农业科学院 Porcine epidemic diarrhea virus pseudorabies virus vector recombinant virus, preparation method and application thereof
CN112626038A (en) * 2021-02-04 2021-04-09 福建省农业科学院畜牧兽医研究所 Construction of pseudorabies virus FB strain gE/gI gene deletion strain and application thereof as vaccine
CN113862230A (en) * 2021-09-30 2021-12-31 中牧实业股份有限公司 Porcine pseudorabies virus gene deletion strain, vaccine composition, preparation method and application thereof

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CN110029079A (en) * 2019-03-27 2019-07-19 南京农业大学 The recombined bacillus subtilis in expression secretion porcine pseudorabies toxalbumin dominant antigen area and application
CN110387354A (en) * 2019-08-16 2019-10-29 江苏省农业科学院 Pseudorabies virus Attenuation strain and its application
CN111154917A (en) * 2020-02-22 2020-05-15 南京农业大学 PCR primer and method for identifying porcine pseudorabies virus variant strain
CN111690619A (en) * 2020-04-20 2020-09-22 华南农业大学 Recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes and construction and application thereof
CN112430625A (en) * 2020-11-23 2021-03-02 武汉枢密脑科学技术有限公司 Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gD protein gene, virus, preparation method and application thereof
CN112430625B (en) * 2020-11-23 2022-08-05 武汉枢密脑科学技术有限公司 Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gD protein gene, virus, preparation method and application thereof
CN112481223A (en) * 2020-12-01 2021-03-12 江苏省农业科学院 Porcine epidemic diarrhea virus pseudorabies virus vector recombinant virus, preparation method and application thereof
CN112626038A (en) * 2021-02-04 2021-04-09 福建省农业科学院畜牧兽医研究所 Construction of pseudorabies virus FB strain gE/gI gene deletion strain and application thereof as vaccine
CN113862230A (en) * 2021-09-30 2021-12-31 中牧实业股份有限公司 Porcine pseudorabies virus gene deletion strain, vaccine composition, preparation method and application thereof
CN113862230B (en) * 2021-09-30 2023-08-08 中牧实业股份有限公司 Porcine pseudorabies virus gene deletion strain, vaccine composition, preparation method and application thereof

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