CN106939320A

CN106939320A - A kind of 2012 plants of infective cloned plasmids of Pseudorabies virus JS, construction method and application

Info

Publication number: CN106939320A
Application number: CN201710118726.9A
Authority: CN
Inventors: 童光志; 郑浩; 王涛; 童武; 李国新; 高飞
Original assignee: Shanghai Veterinary Research Institute CAAS
Current assignee: Shanghai Veterinary Research Institute CAAS
Priority date: 2017-03-01
Filing date: 2017-03-01
Publication date: 2017-07-11
Anticipated expiration: 2037-03-01
Also published as: CN106939320B

Abstract

The present invention provides a pseudorabies virus JS-2012 strain infectious cloning plasmid, construction method and application, based on the newly emerging PRV mutant strain JS-2012 strain in my country, using the loxp-Cre enzyme system to generate the gG of JS-2012 strain The pBeloBAC11 vector sequence was inserted downstream of the gene stop codon, and the infectious cloning plasmid of JS-2012 strain was established, and the genetic sequence operation was carried out by using the galk positive and negative screening system, and the reverse genetic operating system of the mutant strain JS-2012 strain was established. In-depth research on the genetic variation mechanism, virulence enhancement, virus latent infection mechanism and vaccine development of PRV mutant strains provides a technical platform.

Description

A kind of Pseudorabies virus JS-2012 plants of infective cloned plasmids, construction method and application

Technical field

The present invention relates to technical field of bioengineering, more particularly to a kind of Pseudorabies virus infective cloned plasmids and its system Preparation Method and application.

Background technology

Pseudoabies be by caused by Pseudorabies virus (Pseudorabies virus, PRV) to generate heat, very itch, brain ridge Marrow inflammation is a kind of important zoonosis of principal character.PRV can infect a variety of hosts, but pig is the main natural place of the virus Main, storage person and disseminator.The pig of different age group can all infect, and mainly cause in-pig miscarriage, stillborn foetus, mummy tire, Suckling pig high mortality, and boar infertility.PRV belongs to herpetoviridae A type herpesviral subfamilies, and its genome is wire Double-stranded DNA, is about 150kb, is made up of long distinct zones (UL), short distinct zones (US) and US both sides repetitive sequence, coding 70 Multiple protein.Some important viral genes, such as gE, gI, gG and tk, exist on virus virulence and significantly affect, its missing can make Virus weakening.

Pseudo- mad dog betides the U.S. earliest, and China also occurs in that the viral prevalence in early 1960s in swinery, So far PRV is still widely current in China, seriously threatens the health of China swinery, and causes serious financial consequences.PRV infects Pig, in addition to high death is presented in suckling pig, other pigs can resistance to mistake.But in the resistance to peripheral nerve cell for crossing pig, there is PRV's Latent infection.The PRV of latent infection can be activated under certain condition, so that as the infection sources, which increase the difficulty of PRV preventing and treatings Degree.In order to control PRV, China pig farm widely uses PRV attenuated live vaccines, such as PRV Bartha plants epidemic diseases living since the last century end Seedling, the pseudorabies incidence of disease is decreased obviously.But since 2011, the immune large-scale pig farm of many use PRV live vaccines goes out Sow is showed and has produced weak son, stillborn foetus, miscarriage, the clinical symptoms of the pseudo- mad dog such as nervous symptoms and death has occurred in piglet.The country is multiple to grind Group is studied carefully it has proven convenient that the outburst of China's this time porcine pseudorabies is due to caused by China occurs in that Pseudorabies virus variant.With Classical PRV strains SC is compared, and Pseudorabies virus variant virulence strengthens, and Bartha vaccines can not be protected completely to variant Shield.For effective prevention and control PRV variants, it is necessary to which furtheing investigate the hereditary variation of virus and virulence strengthens mechanism.

The content of the invention

The present invention is based on JS-2012 plants of China's emerging PRV variants, using loxp-Cre enzyme systems, in JS- PBeloBAC11 carrier sequences are inserted in 2012 plants of gG gene end codons downstreams, establish JS-2012 plants of infection clones matter Grain, and genetic sequence operation is carried out to it using galk positive and reverse screenings system, establish variant JS-2012 strain reverse genetics behaviour Make system, to further investigate hereditary variation mechanism, virulence enhancing, virus lays dormant infection mechanism and the vaccine development of PRV variants Deng offer technology platform.

1st, by methods of homologous recombination, in JS-2012 plants of gG gene ORF terminator codons downstream insertions two of Pseudorabies virus The wing contains loxp EGFP expression cassettes, obtains recombinant virus rJS2012-gG/EGFP；Using Cre enzymes and loxp sites, delete EGFP expression cassettes, obtain the recombinant virus rJS2012-gG/loxp that loxp is inserted in gG gene ORF terminator codons downstream

2nd, using Cre-loxp, the pBeloBAC11 of EGFP expression cassettes will be inserted between BamHI-HindIII restriction enzyme sites Carrier is recombined into rJS2012-gG/loxp, is obtained in recombinant virus rJS2012-BAC, the recombinant virus, and pBeloBAC11 is carried Body sequence is located at gG downstream of gene.

3rd, by the genomic DNA electricity conversion DH10B bacteriums during rJS2012-BAC infection cells, Orbivirus gene It containing pBeloBAC11, can breed in bacterium, by screening, obtain the plasmid pBAC-JS2012 of viral genomic clone. PBAC-JS2012 transfectional cells, can produce infectious virus resJS-BAC, show JS-2012 pnca gene group cloned plasmids PBAC-JS2012 has infectivity.Using Cre enzymes, the pBeloBAC11 sequences in resJS-BAC can be deleted, resJS- is obtained ΔBAC.ResJS- Δs BAC has and JS-2012 plants of similar growth characteristics of cultured of parental virus and mouse virulence.

4th, by galk screening-genes, gE/gI genes are deleted in pBAC-JS2012 infection clones, and save out GE/gI gene delections virus resJS- Δs gE/gI.

Invention also provides a kind of Pseudorabies virus infection clones, described Pseudorabies virus infection clones are It is prepared by the following method what is obtained：

1) by methods of homologous recombination, in Pseudorabies virus gG gene ORF terminator codons downstream, insertion both wings contain Loxp EGFP expression cassettes, obtain recombinant virus rJS2012-gG/EGFP；Using Cre enzymes and loxp sites, EGFP expression is deleted Frame, obtains the recombinant virus rJS2012-gG/loxp that loxp is inserted in gG gene ORF terminator codons downstream；

2) Cre-loxp is utilized, the pBeloBAC11 of EGFP expression cassettes will be inserted between BamHI-HindIII restriction enzyme sites Carrier is recombined into rPRV-gG/loxp, is obtained in recombinant virus rJS2012-BAC, the recombinant virus, pBeloBAC11 carriers Sequence is located at gG downstream of gene；

3) by the genomic DNA electricity conversion DH10B bacteriums during rJS2012-BAC infection cells, Orbivirus gene It containing pBeloBAC11, can breed in bacterium, by screening, obtain the plasmid pBAC-JS2012 of viral genomic clone；

It is preferred that, wherein described Pseudorabies virus is selected from JS-2012 plants of PRV variants.

Meanwhile, the present invention also provides a kind of utilization Pseudorabies virus infection clones and is preparing the examination of detection Pseudorabies virus Purposes in agent or medicine；

Meanwhile, the present invention also provides a kind of Pseudorabies virus infection clones and is preparing prevention or treatment pseudoabies viral disease Purposes in biological products；

Meanwhile, the present invention also provides a kind of construction method for the Pseudorabies virus infection clones for preparing gE/gI missings, institute The method of stating comprises the following steps：

1) the plasmid pBAC-JS2012 of viral genomic clone, is obtained；

2), using galk positive and reverse screening systems, gE/gI genes is lacked on PRV infection clones pBAC-JS2012, are obtained Obtain the Pseudorabies virus infection clones pBACJS- Δs gE/gI of gE/gI missings；

It is preferred that, wherein described Pseudorabies virus is selected from JS-2012 plants of PRV variants；

Meanwhile, the present invention also provides the virus that a variety of utilization Pseudorabies virus infection clones are saved out, including but does not limit In：resJS-BAC、resJS-ΔBAC、resJS-ΔgE/gI.

Meanwhile, the present invention also provides a kind of Pseudorabies virus infection clones of differentiation gE/gI missings and contaminated with natural sense The detection method of strain, comprises the following steps：Enter performing PCR using LgEgIF/RgEgIR for primer to identify, it is pseudo- mad that gE/gI is lacked Dog virus infection clones amplify 1000bp band, are positive bacterium colony, and the natural infection strain of the non-missings of gE/gI is expanded Bp bands more than 2000.

It should be noted that this method is not just in JS-2012 plants of operation, can apply to many plants of PRV diseases Strain, those skilled in the art can be by conventional experimental implementation and molecular biology manipulations, in technical solution of the present invention Under enlightenment, the corresponding operation of other strains is realized.

Brief description of the drawings

Examples of the present invention will be described by way of reference to the accompanying drawings, wherein：

Fig. 1 is, using PRV JS-2012 genomes as template, to be expanded respectively using primer PgGF/PgGR and PDgGF/PDgGR Left and right sides homology arm, PCR amplification electrophoretograms, wherein, 1：DNA Marker DL2000,2：Left restructuring arm, 3：Right restructuring Arm；

Fig. 2 is that 2ug pTEGGF are transfected into BHK-21 cell green fluorescence figures using the transfection reagents of FuGENE 6；

Fig. 3 is to use the transfection reagents of FuGENE 6 by 2ug PRV JS-2012 genomes and 1ug pTEGGF plasmid corotation Contaminate BHK-21 cell green fluorescence figures；

Fig. 4 is recombinant virus rJS2012-gG/loxp PCR amplification qualification result electrophoretograms；

Fig. 5 is recombinant virus rJS2012-gG/loxp PCR amplification qualification result sequence analysis figures；

Fig. 6 is expanded using the primer PEGFPF/PEGFPR PCR for expanding EGFP expression cassettes and identified using pEGFP-C3D as template As a result electrophoretogram, wherein, 1：DNA Marker DL2000,2：Pcr amplified fragment；

Fig. 7 is that 2ug pBeloBAC11-EGFP are transfected into BHK-21 cell green fluorescences using the transfection reagents of FuGENE 6 Figure；

Fig. 8 is to use the transfection reagents of FuGENE 6 by 1ug pBeloBAC11-EGFP, 1ug pcDNA3.1-cre, 2ug RJS2012-gG/loxp genome cotransfection BHK-21 cell green fluorescence figures；

Fig. 9 is to carry out restricted enzyme cutting spectrum analysis figure to 3 cloned plasmids using BamHI, wherein, 1：DNA Marker DL15000；2：Genomic clone plasmid 1；3：Genomic clone plasmid 2；4：Genomic clone plasmid 3；

Figure 10 is to use the transfection reagents of FuGENE 6 by 2 μ g pBAC-JS2012 transfected Vero cells green fluorescence figures；

Figure 11 is PRV JS-2012, resJS-BAC, resJS- Δs BAC viral one step growth curve figure；

Figure 12 is PRV JS-2012, resJS-BAC, resJS- Δs BAC virus plaque lab diagram；

Figure 13 is survival rate after mouse inoculation JS-2012 and resJS- Δ BAC.Wherein, (A) 10⁴TCID₅₀Inoculation；(B) 10⁵TCID₅₀Inoculation；

Figure 14 is, using plasmid pGalk as template, to expand galk expression cassettes PCR using primer galkup/galkdown and expand As a result electrophoretogram, wherein, 1：DNA Marker DL2000,2：PCR primer；

Figure 15 be using bacterium solution be template, PgBDF/PgBDR and LgalkDF/LgalkDR be primer enter performing PCR screening PCR Amplification electrophoretogram, wherein, 1：DNA Marker DL2000,2-7：PgBDF/PgBD qualification results, 8-13：LgalkDF/ LgalkDR qualification results；

Figure 16 is, using pBAC-JS2012 as template, to be expanded using primer LgEgIF/LgEgIR and RgEgIF/RgEgIR left Homology arm PCR amplification electrophoretograms, wherein, 1：DNA Marker DL2000,2：Left homology arm；

Figure 17 is, using pBAC-JS2012 as template, to be expanded using primer LgEgIF/LgEgIR and RgEgIF/RgEgIR right Homology arm PCR amplification electrophoretograms, wherein, 1：DNA Marker DL2000,2：Right homology arm；

Figure 18 is to screen transfer vector electrophoretogram so that the identification of XhoI and BamHI double digestions is negative, wherein, 1：DNA Marker DL5000,2：pSKDEI-1、3：pSKDEI-2；

Figure 19 is to enter performing PCR identification electrophoretogram using LgEgIF/RgEgIR for primer, wherein, M1：DNA Marker DL5000,1-21：Detect sample, c：PJS2012-BAC is compared, M2：DNA Marker DL2000；

Figure 20 is with resBACJS- Δ gE/gI vero cells infection green fluorescence figures；

Figure 21 is to extract resJS- Δ gE/gI genomes, is identified with LgEgIF/RgEgIRPCR, sequencing result figure；

Figure 22 is the expression that the gE albumen in resJS- Δ gE/gI infection cells is detected with IFA, wherein, A：PRV JS- 2012, B：ResJS- Δs gE/gI, C：negative control；

Figure 23 is resJS- Δs gE/gI and parent plant JS-2012 one step growth curve figure；

Figure 24 is resJS- Δs gE/gI and parent plant JS-2012 plaque aspect graph；

Embodiment

In the following example, the experimental method of unreceipted actual conditions, generally routinely condition, such as《Fine works molecular biosciences Learn experiment guide》(Ma Xuejun, Su Yuelong's translates Beijing by F.M. Ao Sibai, R.E. James Kingstons, the chief editor such as J.G. Sai Deman：Section Learn publishing house, 2004) described in method carry out.

The present invention, by homologous recombination, is terminated on the basis of Pseudorabies virus variant JS-2012 in viral genome gG Codon downstream introduces EGFP marker gene and loxp sites, and is deleted using Cre enzymes/loxp recombination system in recombinant virus EGFP gene, obtain rJS2012-gG/loxp.In the recombination system using Cre enzymes/loxp, the BAC of EGFP gene will be carried Carrier is recombined into rJS2012-gG/loxp, obtains recombinant virus rJS2012-BAC.During rJS2012-BAC infection cells Genomic DNA electricity conversion DH10B bacteriums, screening obtain viral genomic clone plasmid pBAC-JS2012.

PBAC-JS2012 transfectional cells, obtain infective viral resJS-BAC, it was demonstrated that pBAC-JS2012 has infection Property.Viral resJS- Δs BAC using BAC carrier sequence in Cre enzymes/loxp system-kills resJS-BAC has and parent plant Close JS-2012 growth characteristics and to mouse strong virus force feature.Using galk positive and reverse screening systems, in pBAC-JS2012 The clone pBACJS- Δ gE/gI of gE/gI missings are successfully constructed, gE is not expressed from the viral resJS- Δs gE/gI of Clone Origin Albumen, it was confirmed that pBAC-JS2012 operability.Infectious gram of the PRV variants JS-2012 that the present invention is successfully constructed Grand pBAC-JS2012, and its operating method is established, to utilize reverse genetics research virus replication, latent infection and variation Strain virulence enhancing mechanism, the new attenuated vaccine of exploitation and structure viral vectors etc. are all significant.

Below by embodiment, the present invention is specifically described.

In following embodiments of the present invention, the major experimental material used is as follows：

Virus and cell：(American Cell collection ATCC, preserving number is BHK-21 cells：CCL-10), Vero cells (American Cell collection ATCC, preserving number is：CCL-81), (this laboratory is preserved Pseudorabies virus variant JS-2012； Publish an article and see：Separation and the Chinese animals of identification of Pseudorabies virus in sequela piglet is immunized in the such as Tong Wu, Zhang Qingzhan, Zheng Hao Infectious disease journal, 2013,21 (3)：1-7).

Plasmid and bacterial strain：PBeloBAC11 carriers are ground with Escherichia coli DH10B by Chinese Academy of Agricultural Sciences Harbin animal doctor Study carefully institute Wang Yunfeng researcher to give, pgalk plasmids are given with Escherichia coli SW102 by He'nan University professor Cao Gengsheng, PcDNA3.1-cre is purchased from and first biotechnology (Shanghai) limited company, and pEGFP-N1, pEGFP-C3 are purchased from Clontech Company, pMD18-T, bacillus coli DH 5 alpha are purchased from Takara companies.

Other reagents：Restriction enzyme and T4DNA ligases are purchased from NEB companies, and the transfection reagents of FuGENE 6 are purchased from Promega companies, hyclone (fetal bovine serum, FBS), MEM, DMEM are purchased from Gibco companies, and plasmid is small to propose examination Agent box is purchased from extraction reagent kit in QIAGEN companies, plasmid and is purchased from Macherey-Nagel companies, and gel reclaims kit is purchased from wide State Dongsheng bio tech ltd, PCR reagent is purchased from Takara companies.Reagent needed for positive and reverse screening is purchased from raw work bioengineering (Shanghai) limited company.

In embodiments of the invention, the cultural method of BHK-21 cells and Vero cells is as follows：

BHK-21 cells in the T25 Tissue Culture Flasks of the MEM culture mediums containing 10%FBS it is adherent cover with individual layer after, abandon Culture medium in Tissue Culture Flask is removed, and is washed once with PBS, new contain under cell dissociation, will be added with 0.25% pancreatin 10%FBS MEM culture mediums, with 1: 8 ratio point kind in cell bottle or Tissue Culture Plate.

Vero cells in the T25 Tissue Culture Flasks of the DMEM culture mediums containing 10%FBS it is adherent cover with individual layer after, discard Culture medium in Tissue Culture Flask, and being washed once with PBS, under cell dissociation, will be added with 0.25% pancreatin and new contains 10% FBS DMEM culture mediums, with 1: 5 ratio point kind in cell bottle or Tissue Culture Plate.

The recombinant virus rJS2012-gG/loxp of embodiment 1 structure

In order to build JS-2012 plants of infection clones of Pseudorabies virus, the present embodiment, will by the method for homologous recombination In loxp sequences and marker gene EGFP insertion JS-2012 genomes, recombinant virus rJS2012-gG/EGFP is obtained.Pass through again Cre enzyme effects, delete the EGFP marker gene in rJS2012-gG/EGFP, obtain the recombinant virus rJS2012- for carrying loxp gG/loxp。

Specific implementation process is as follows：

1.1 transfer vector pTEGGF structure

First by XhoI and BamHI digestion pEGFP-C3, and with T4DNA polymerase filling-in, glue reclaim large fragment And with T4DNA Ligase connections, obtain and delete XhoI to the plasmid pPEGFP-C3D of multiple cloning sites sequence between BamHI.With PRV JS-2012 genomes are template, expand left and right respectively using primer PgGF/PgGR (table 1) and PDgGF/PDgGR (table 1) Side homology arm, PCR amplifications such as Fig. 1 (in Fig. 1,1：DNA Marker DL2000,2：Left restructuring arm, 3：Right restructuring arm), and Amplified production is connected into pMD18-T, recombinant plasmid pGL-ARM and pGR-ARM is obtained.Utilize SalI and AseI digestions pGL- ARM, AseI and XbaI enzyme cutting pEGFP-C3D, reclaim small fragment, is connected with T4DNA Ligase into through SalI and XbaI enzyme cutting PBlueScript SK (+), obtain recombinant plasmid pLARM-EGFP.With AflII and EcoRI difference digestion pGR-ARM and PEGFP-N1, will connect into pGR-ARM from the pEGFP-N1 polyA fragments cut, obtains recombinant plasmid pRARM-PA.With XhoI With XbaI enzyme cutting pRARM-PA, small fragment is reclaimed, the pLARM-EGFP through same enzyme digestion is connected into T4DNA Ligase, obtained Transfer vector pTEGGF.2ug pTEGGF are transfected into BHK-21 cells using the transfection reagents of FuGENE 6,30h after transfection turns There is green fluorescence expression (Fig. 2) in dye cell.All transfection methods of this explanation enter according to the transfection reagent specifications of FuGENE 6 Row operation.

Pcr amplification primer thing summary sheet in 1 explanation of table

1.2 recombinant virus rJS2012-gG/EGFP

BHK-21 cells are infected with PRV JS-2012,20h after infection, harvesting poison extracts JS- with phenol-chloroform method 2012 genomic DNAs.With the transfection reagents of FuGENE 6 by 2ug PRV JS-2012 genomes and 1ug pTEGGF plasmid co-transfections BHK-21 cells.36h after transfection, transfectional cell has the cytopathy (Fig. 3) of green fluorescence expression.Treat that 60% cell occurs Lesion, receives supernatant progress Plaque-purified.The single virus plaque for having green fluorescence by continuous 3 wheel pickings is purified, and is obtained Respectively there are the recombinant virus of a loxp sites insertion, referred to as rJS2012-gG/EGFP containing EGFP and its both wings.

1.3 recombinant virus rJS2012-gG/loxp

2ug pcDNA3.1-cre are transfected into BHK-21 cells using the transfection reagents of FuGENE 6.10h after transfection, with 0.01MOI recombinant viruses rJS2012-gG/EGFP is infected.70% cytopathy to appear, receives supernatant progress Plaque-purified. Picking does not have the single virus plaque of green fluorescence, obtains the recombinant virus for not expressing EGFP, referred to as rJS2012-gG/ loxp.And whether delete EGFP using primer gLoxF/gLoxR (table 1) detection recombinant viruses and contain single loxp sites.PCR Reaction system：94 DEG C of 5min, 94 DEG C of 30s, 68 DEG C of 1min, 68 DEG C of 2min30s, carry out 30 circulations altogether；72℃ 10min. PCR primer electrophoresis result such as Fig. 4 (1：DNA Marker DL2000,2：Pcr amplified fragment), amplify 1 treaty 550bp bar Band, it is in the same size with expection.Purpose band is sequenced, sequence is shown in SEQ ID in specification nucleotides and amino acid sequence table NO.21, sequence analysis (Fig. 5) display, 34bp loxp sequences are successively inserted into PRV gG Orf downstreams.

The recombinant virus rJS2012-BAC of embodiment 2 structure

The present embodiment, by means of the loxp sites in rJS2012-gG/loxp, is marked by loxp-Cre enzyme systems by carrying Note gene EGFP bacterial artificial chromosome carrier is recombined into rJS2012-gG/loxp, obtains recombinant virus rJS2012-BAC. Specific implementation process is as follows：

2.1 transfer vector pBeloBAC11-EGFP structure

Using pEGFP-C3D as template, EGFP expression cassettes, PCR reactants are expanded using primer PEGFPF/PEGFPR (table 1) System：94 DEG C of 5min, 94 DEG C of 30s, 57 DEG C of 45s, 72 DEG C of 2min, carry out 30 circulations altogether；72℃ 10min.PCR primer electricity Swim result such as Fig. 6 (in Fig. 6,1：DNA Marker DL2000,2：Pcr amplified fragment), amplify 1600bp or so purpose bars Band., will with T4DNA ligases through glue reclaim with BamHI and HindIII difference digestion target DNA bands and pBeloBAC11 EGFP expression cassettes are connected with pBeloBAC11.Connection product converts DH5 α, with BamHI and HindIII digestion screening positive clones, Obtain transfer vector pBeloBAC11-EGFP.2ug pBeloBAC11-EGFP are transfected into BHK- using the transfection reagents of FuGENE 6 There is green fluorescence expression (Fig. 7) in 36h after 21 cells, transfection, transfectional cell.

2.2 recombinant virus rJS2012-BAC

According to the method for embodiment 1.2, rJS2012-gG/loxp genomic DNAs are extracted.Will with the transfection reagents of FuGENE 6 1ug pBeloBAC11-EGFP, 1ug pcDNA3.1-cre, 2ug rJS2012-gG/loxp genome cotransfections BHK-21 are thin Born of the same parents.After cotransfection 36h, it is seen that the expression (Fig. 8) of lesion and green fluorescence.70% cytopathy is treated, supernatant progress plaque is received pure Change.Picking has the single virus plaque of green fluorescence, carries out the recombinant virus that purifying obtains BAC carrier insertion, name rJS2012-BAC。

The structure of embodiment 3PRV infectious bacteria artificial chromosomes

The present embodiment by rJS2012-BAC genome electricity by being transformed into Escherichia coli DH10B, and screening, which is obtained, has infection The plasmid pBAC-JS2012 of property.Specific implementation process is as follows：

Vero cells are infected with rJS2012-BAC, 16-21h after infection extracts rJS2012- in cell using Hirt methods Ring-type genome in BAC reproduction processes, electricity conversion Escherichia coli DH10B, electric conversion condition is as follows：1mm electricity conversion cups, 1750V, 25 μ F, 200 Ω, electric shock is once.0.8mL S.O.C nutrient solutions are added after electric shock immediately, and electric transformed bacteria is moved into 2.0mL centrifuge tubes, 37 DEG C of culture 1h, 5000rmp centrifugation 2min, are coated on the LB containing chloramphenicol (25 μ g/mL) by precipitation bacterium and put down Plate, 37 DEG C of culture 24h.Bacterial clump on picking flat board, puts in the LB fluid nutrient mediums containing chloramphenicol (25 μ g/mL), 37 DEG C culture 15h, extract restructuring BAC plasmids.Respectively using PgBDF/PgBDR and PgCDF/gCDR as primer pair, PCR detection restructuring Whether BAC plasmids, identification contains gB and gC genes.Screening obtains 3 cloned plasmids containing gB, gC gene.Using BamHI to 3 Individual cloned plasmids carry out restricted enzyme cutting spectrum analysis, digestion band electrophoresis result such as Fig. 9 (1：DNA Marker DL15000； 2：Genomic clone plasmid 1；3：Genomic clone plasmid 2；4：Genomic clone plasmid 3), three cloned plasmids BamHI digestions Collection of illustrative plates is close, more than 15kb bands 2,3 between 7.5kb-10kb, 4 between 5kb-7.5kb, 4,2.5kb between 2.5kb-5kb 1 below.1 band between the difference of one band of presence between cloned plasmids 1 and cloned plasmids 2,3, i.e. 2.5kb-5kb, Cloned plasmids 1 are bigger than between cloned plasmids 2,3.Bam HI restriction enzyme sites in JS-2012 genome sequences are analyzed, prediction Big digestion band is consistent with electrophoresis pattern.Cloned plasmids 3 are referred to as pBAC-JS2012 (SEQ ID NO.24), selection is carried out Follow-up test.

Using the transfection reagents of FuGENE 6 by 2 μ g pBAC-JS2012 transfected Vero cells, 24h after transfection, cell occurs The cytopathy (Figure 10) of green fluorescence is expressed, treats that cytopathy, up to 70%, harvests supernatant, obtains Revive virus resJS-BAC. It is above-mentioned test result indicate that, pBAC-JS2012 transfectional cells can produce infectious viral particle, with infectivity.JS-2012 plants Infection clones pBAC-JS2012 is successfully constructed.

With reference to embodiment 1.3, with the transfection reagents of FuGENE 6 by 2ug pcDNA3.1-cre transfected Vero cells, transfect 10h, is infected with 0.01MOI recombinant viruses resJS-BAC afterwards.70% cytopathy to appear, receives supernatant progress plaque pure Change.The single plaque for selecting redgreen fluorescent protein expression by three-wheel is purified, and is obtained without BAC carrier sequence PRV saves strain, referred to as resJS- Δs BAC.With reference to the method for embodiment 1.3, by template of resJS- Δ BAC genomes, GLoxF/gLoxR is primer, and the sequencing fragment amplified is shown, sequence is consistent with rJS2012-gG/loxp sequence.This table Bright, BAC carrier sequence can be deleted in Revive virus resJS-BAC, the viral resJS- Δs BAC and recombinant virus of acquisition RJS2012-gG/loxp has identical hereditary feature.

The Revive virus growth characteristics of embodiment 4 are analyzed

By this embodiment, Revive virus resJS- Δs BAC growth kinetics feature, plaque form and JS- is found 2012 plants similar, and mouse challenge viral dosage shows that resJS- Δs BAC is identical with JS-2012 plants of virulence, and this display is from infectious gram The grand middle viral resJS- Δs BAC saved out has and parent plant identical biological characteristics.

4.1 one step growth curves

PRV JS-2012, resJS-BAC, resJS- Δs BAC is inoculated with respectively with 1MOI and covers with Vero cell monolayer In T25, be incubated 1h after, washed one time with DMEM, in each blake bottle plus 5ml 2%FBS DMEM inoculation after respectively with inoculation after Culture supernatant 500ul is taken respectively at 4,8,12,16,20,24,28,32,36,40h, and adds DMEMs of the 500ul containing 2%FBS. The virus of different time points is diluted to 10 with 1: 10^-1To 10^-9Dilution factor, and be inoculated into and be grown on 96 porocyte culture plates In Vero cell monolayer, each dilution factor connects 8 holes.After inoculating cell culture 4d, cytopathy of the observation per hole, according to Reed-Muench methods calculate virus TCID50, utilize the viral one step growth curve of GraphPad Software on Drawing.Viral growth curves Such as Figure 11, three strain virus have similar growth kinetics characteristic, but Revive virus resJS-BAC, resJS- Δ BAC highest Titre is more slightly lower than parent's strain JS-2012.

4.2 plaque assays

Revive virus resJS-BAC, resJS- Δ BAC and parent poison PRV JS-2012 are used into DMEM culture mediums respectively After dilution, Vero cells are inoculated with, after being incubated 1 hour, virus liquid are discarded, PBS is washed twice, 3ml is added per hole and contains 1% low melting point The covering culture medium of agarose and 2%FBS.After room temperature solidification, culture 3d is inverted in 37 DEG C, 5%CO2, with 5% crystal violet Dyeing, as a result such as Figure 12.In fig. 12, Revive virus resJS- Δs BAC and parent poison PRV JS-2012 after BAC carrier are deleted Plaque form is consistent, and size is close, and the resJS-BAC of the sequence containing BAC is less than normal than JS-2012.

The pathogenic experiment of 4.3 mouse

Respectively with 10⁴TCID₅₀/ only with 10⁵TCID₅₀/ only PRV JS-2012 and resJS- Δ BAC subcutaneous abdomens inoculation 7 SPF grades of Kunming female mices of week old, 5 groups, are compareed totally containing one group by inoculum of DMEM by every group 10.Observation mouse is dead daily Situation is died, mouse survival curve is drawn.As a result such as Figure 13,10⁴TCID₅₀It is inoculated with (13A), resJS- Δs BAC survival rate and dead Die the time and PRV JS-2012 are completely the same；10⁵TCID₅₀It is inoculated with (13B), resJS- Δs BAC and PRV JS-2012 inoculation groups Completely dead, the death time concentrates on 4d and 5d after inoculation, but resJS- Δs BAC is slightly higher in the 4d death rates.This shows, Revive virus is deleted after BAC sequences, maintains the strong malicious features of parent plant JS-2012.

Embodiment 5 builds gE/gI deleted virus using galk positive and reverse screening system operatios pJS2012-BAC

The present embodiment utilizes galk positive and reverse screening systems, and gE/gI bases are lacked on PRV infection clones pBAC-JS2012 Cause, constructs gE/gI deleted virus resJS2012- Δ gE/gI, so as to establish JS-2012 infection clones pBAC- JS2012 operating method.Specific implementation process is as follows：

5.1 positive screenings

With reference to electric method for transformation in embodiment 3, pBAC-JS2012 plasmid electricity is rotated into SW102 bacterial strains, contained PBAC-JS2012 bacterial strains SW102-BAC.

Using plasmid pGalk as template, galk expression cassettes, PCR reactions are expanded using primer galkup/galkdown (table 1) System：94 DEG C of 4min, 94 DEG C of 1Ss, 60 DEG C of 30s, 72 DEG C of 1min, carry out 30 circulations altogether；72℃ 10min.PCR primer Electrophoresis result such as Figure 14 (1：DNA Marker DL2000,2：PCR primer), amplification obtains 1300bp or so purpose band, glue Reclaim the band.

The electric transformed competence colibacillus of SW102-BAC bacterial strains is prepared, according to electric method for transformation in embodiment 3, galk bands are turned Enter SW102-BAC.Transformed bacteria is applied into M63 flat boards, and (15g Agar are dissolved in the sterilizing of 800ml deionized waters mesohigh, then add 200ml5xM63,1ml 1M MgSO4,10ml 20% galactolipin, 5ml 0.2mg/ml biotins, 4.5ml 10mg/ml L- are bright After propylhomoserin, 500ul chloramphenicol (25mg/ml), sterilized petri dishes, obtain M63 flat boards) just screened.After 32 DEG C of culture 3d, choose Take bacterium colony to cultivate 16h in the LB liquid containing chloramphenicol (25 μ g/ml), directly using bacterium solution be template, PgBDF/PgBDR with LgalkDF/LgalkDR (table 1) enters performing PCR screening gB and galk positive colonies, PCR amplifications such as Figure 15 (1 for primer：DNA Marker DL2000,2-7：PgBDF/PgBD qualification results, 8-13：LgalkDF/LgalkDR qualification results), 6 detected Individual clone, can amplify gB genetic fragments, but wherein only have 3 clones to detect galk genes, and size is consistent with expection. Because LgalkDF is located at galk recombination sites upstream in JS-2012 genomes, LgalkDR is located in galk genes, LgalkDF/ LgalkDR amplifies fragment, then shows in galk genetic recombination such as viral genome.Therefore, three amplify fragment clone For positive colony, SW102-BAC-galk+ is named.Confirm galk sequence as shown in SEQ ID NO.23 after sequencing.

5.2 counter-selections

Using pBAC-JS2012 as template, expanded using primer LgEgIF/LgEgIR and RgEgIF/RgEgIR left and right homologous Arm, left homology arm fragment such as Figure 16 (1：DNA Marker DL2000,2：Left homology arm), right homology arm fragment such as Figure 17 (1： DNA Marker DL2000,2：Right homology arm), a length of 500bp of two homology arms or so.The left homology arm of glue reclaim, right homology arm and Eco RV digestion pBluescriptSK carriers, using vitro recombination kit Gibson Assembly Master Mix by it is left, Right restructuring arm is connected with carrier, obtains negative screening transfer vector pSKDEI.With the negative screening transfer of XhoI and BamHI double digestions identification Carrier, such as Figure 18 (1：DNA Marker DL5000,2：pSKDEI-1、3：PSKDEI-2), can be by pSKDEI digestions into 1000bp With 3000bp or so two bands, it is consistent with sequence analysis profiling results.

Using pSKDEI as template, with LgEgIF/RgEgIR (table 1) for primer, PCR amplifications obtain DNA fragmentation DgE/gI.Ginseng According to the method for embodiment 3, DgE/gI fragment electricity is transferred in SW102-BAC-galk+, transformed bacteria is applied into M63 bears screening flat board (15g Agar are dissolved in the sterilizing of 800ml deionized waters mesohigh, then add 200ml 5xM63,1ml 1M MgSO4,10ml 20% The glycerine of DOG, 10ml 20%, 5ml 0.2mg/ml biotins, 4.5ml 10mg/ml L-Leus, 500ul chloramphenicol (25mg/ Ml after), sterilized petri dishes, obtain M63 and bear screening flat board).After 32 DEG C of culture 3d, picking colony is in containing chloramphenicol (25 μ g/ml) 32 DEG C of LB liquid culture 16h, enter performing PCR for primer using LgEgIF/RgEgIR and identify.PCR primer electrophoresis result such as Figure 19 (M1：DNA Marker DL5000,1-21：Detect sample, c：PBAC-JS2012 is compared, M2：DNA Marker DL2000), 15 bacterium colony cultures amplify 1000bp band, are positive bacterium colony, and the pBAC- of the non-missings of gE/gI JS2012 expands bp bands more than 2000.Positive colony is referred to as pBACJS- Δs gE/gI (SEQ ID NO.25).

5.3resJS- Δ gE/gI virus rescues

PBACJS- Δ gE/gI are extracted from positive colony bacterium solution using NucleoBond Xtra Midi kits, to turn 2ug pBACJS- Δ gE/gI transfected Vero cells are obtained virus resBACJS- Δs gE/gI by transfection reagent FuGENE 6.With It may occur in which that green fluorescence expresses (Figure 20) in cell after resBACJS- Δ gE/gI vero cells infections, infection 20h.With reference to implementation The method of example 1.3, using the transfection reagents of FuGENE 6 by 2ug pcDNA3.1-cre transfected Vero cells, then with resBACJS- Δ gE/gI infection cells.Treat that 60% lesion occurs in cell, harvest supernatant, carry out Plaque-purified.Selected by three-wheel without green The plaque of fluorescent protein expression, obtains virus and is referred to as resJS- Δs gE/gI.

5.4resJS- Δs gE/gI is identified

ResJS- Δ gE/gI genomes are extracted, are identified with LgEgIF/RgEgIR (table 1) PCR, sequencing result such as SEQID Shown in NO.22.Compared with JS-2012 genomes, one section of gI gene coding region the 571st is contained in resJS- Δ gE/gI genomes Position nucleotides to 1211 nucleotides in gE gene coding regions missing (Figure 21).

Vero cells in 6 orifice plates are infected with resJS- Δs gE/gI, while using JS-2012 infection as positive control, DMEM Infect for negative control, using this laboratory gE monoclonal antibodies as primary antibody, sheep anti-mouse igg is secondary antibody, existed with IFA detections The expression of gE albumen in resJS- Δ gE/gI infection cells.Such as Figure 22 (A：PRV JS-2012, B：ResJS2012- Δ gE/gI, C：negative control.), only it is presented green fluorescence in street strain JS-2012 infection cells, and resJS- Δs gE/gI It is identical with the negative control cell that DMEM is inoculated with, fluorescence is had no, this shows that resJS- Δs gE/gI does not express gE albumen.

5.5resJS- Δ gE/gI biological property analysis

With reference to the method for embodiment 4.1 and 4.2, compare resJS- Δs gE/gI and parent plant JS-2012 one step growth curve And plaque form.One step growth curve (Figure 23) result shows that resJS- Δ gE/gI growth rates are compared with JS-2012, slightly Decline.Plaque assay (Figure 24) shows that resJS- Δ gE/gI plaques are less than normal than JS-2012.This shows deleting for gE/gI genes Remove, the propagation and iuntercellular that can influence virus spread.

The invention is not limited in foregoing embodiment.The present invention, which is expanded to, any in this manual to be disclosed New feature or any new combination, and disclose any new method or process the step of or any new combination.

Claims

1. a kind of construction method of Pseudorabies virus infection clones, it is characterised in that the described method comprises the following steps：

1) by methods of homologous recombination, in Pseudorabies virus gG gene ORF terminator codons downstream, insertion both wings contain loxp's EGFP expression cassettes, obtain recombinant virus rJS2012-gG/EGFP；Using Cre enzymes and loxp sites, EGFP expression cassettes are deleted, are obtained Obtain the recombinant virus rJS2012-gG/loxp that loxp is inserted in gG gene ORF terminator codons downstream；

2) Cre-loxp is utilized, the pBeloBAC11 carriers of EGFP expression cassettes will be inserted between BamHI-HindIII restriction enzyme sites It is recombined into rJS2012-gG/loxp, obtains in recombinant virus rJS2012-BAC, the recombinant virus, pBeloBAC11 carrier sequences Row are located at gG downstream of gene；

3) by the genomic DNA electricity conversion DH10B bacteriums during rJS2012-BAC infection cells, Orbivirus gene contains PBeloBAC11, can breed in bacterium, by screening, obtain the plasmid pBAC-JS2012 of viral genomic clone.

2. a kind of construction method of Pseudorabies virus infection clones as claimed in claim 1, wherein described pseudoabies Poison is selected from JS-2012 plants of PRV variants.

3. the Pseudorabies virus infection clones that method according to claim 1 or 2 is built.

4. Pseudorabies virus infection clones as claimed in claim 3, it is characterised in that described Pseudorabies virus is selected from JS-2012 plants of PRV variants.

5. the Pseudorabies virus infection clones described in claim 3 or 4 are preparing the reagent or medicine of detection Pseudorabies virus In purposes.

6. the Pseudorabies virus infection clones described in claim 3 or 4 are preparing prevention or treatment Pseudorabies virus diease occurrence thing Purposes in product.

7. a kind of construction method for the Pseudorabies virus infection clones for preparing gE/gI missings, methods described comprises the following steps：

1) the plasmid pBAC-JS2012 of viral genomic clone, is obtained as claimed in claim 1；

2), using galk positive and reverse screening systems, gE/gI genes are lacked on PRV infection clones pBAC-JS2012, gE/ is obtained The Pseudorabies virus infection clones pBACJS- Δs gE/gI of gI missings.

8. a kind of construction method of Pseudorabies virus infection clones for preparing gE/gI missings as claimed in claim 7, wherein Described Pseudorabies virus is selected from JS-2012 plants of PRV variants.

9. the Pseudorabies virus infection clones for the gE/gI missings that method according to claim 8 is built.

10. a kind of the Pseudorabies virus infection clones and natural infection strain of the gE/gI missings distinguished described in claim 8 Detection method, it is characterised in that comprise the following steps：Enter performing PCR using LgEgIF/RgEgIR for primer to identify, gE/gI missings Pseudorabies virus infection clones amplify 1000bp band, be positive bacterium colony, and the natural sense of the non-missings of gE/gI is contaminated Strain expands bp bands more than 2000.