CN109628416A - A kind of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain and its construction method - Google Patents

Abstract

The invention belongs to porcine pseudorabies virus technical field, a kind of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain and its construction method are disclosed.The recombinant porcine pseudorabies Strain is rPRV-gE^‑/TK^‑/ORF2⁺.Recombinant virus rPRV-gE of the present invention^‑/TK^‑/ORF2⁺Can inducing mouse generate the specific antibody for being directed to PCV2, anti-PRV neutralizing antibody titers can reach 1: 64；Illustrate that the recombinant virus has preferable immunogenicity, provides a kind of alternative vaccine strain for two kinds of viral infection of PRV and PCV2 can be prevented simultaneously.

Description

A kind of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain and Its construction method

Technical field

The invention belongs to porcine pseudorabies virus technical fields, and in particular to a kind of expression porcine circovirus 2 type ORF2 base Because of recombinant porcine pseudorabies Strain and its construction method.

Background technique

Pig circular ring virus (porcine circovirus, PCV) belongs to circovirus section (Circoviridae) annulus disease Poison belongs to (Circovirus), is divided into 1 type of circovirus (PCV1) and circovirus 2 type according to gene composition and antigenicity (PCV2) two serotype, PCV1 is to pig there is no pathogenic, and PCV2 then has.PCV2 is that postweaning multisystemic is caused to decline Exhaust the main pathogen of syndrome (PMWS), additionally it is possible to cause pigskin inflammation and nephrotic syndrome (PDNS) and sow breeding difficulty, and Often with a variety of cause of disease mixed infections.The disease is widely current in many countries at present, causes huge economic damage to breeding enterprise It loses.PCV2 full-length genome 1766-1768bp is read containing two main openings of ORF1 (945bp) and ORF2 (702/705bp) Frame.The Cap protein of ORF2 coding is the unique structural proteins of PCV2 virion, constitutes viral capsid, and molecular weight is about 27.8 kDa, the albumen can be the main immunogenic proteins of PCV2 in conjunction with the virus receptor of host cell.

Porcine pseudorabies (Porcine Pseudorabies, PR) are by porcine pseudorabies virus (Pseudorabies Virus, PRV) caused by a kind of acute infectious disease.The disease is generated heat with piglet, encephalomyelitis and pregnant sow miscarriage are main special Disease.Newborn piglet is usually acute infection, there is also the respiratory symptoms such as vomiting, diarrhea, the death rate in addition to having nervous symptoms Almost it is up to 100%.

It is transformed using gene of the molecular biology method to PRV, the vaccine for making it suitable for expression alien gene claims For using PRV as the recombinant vaccine of carrier.PRV has many advantages, such as the carrier of live vaccine: genome is big, is inserted into for foreign gene Site it is more；Host range is wide, and pig, sheep, ox, dog etc. can all infect；Duration of immunity is long, sustainable expression alien gene；Replicate energy Power is strong, and the expression quantity of foreign gene can be improved；More most important to be, which does not threaten the mankind.

Therefore, under the support of the prior art, developing a kind of strain that can effectively prevent pig PCV2 and PR is still Domestic and international worker needs continuous effort and innovation.

Summary of the invention

The purpose of the present invention is to provide a kind of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strains And its construction method.

To achieve the above object, the technical solution adopted by the present invention is as follows:

A kind of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain, the recombinant porcine pseudorabies virus Strain is rPRV-gE^-/TK^-/ORF2⁺。

The construction method of the expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain:

(1), the building of recombinant plasmid pTK-ORF2:

(1.1), the primer of PCR amplification ORF2, CMV-ORF2-poly A, TKU-CMV-ORF2-polyA-TKL are designed, respectively Are as follows:

F1: as shown in SEQ ID No.1；R1: as shown in SEQ ID No.2；

F2: as shown in SEQ ID No.3；R2: as shown in SEQ ID No.4；

F3: as shown in SEQ ID No.5；R3: as shown in SEQ ID No.6；

Wherein: F1 and R1 identifies ORF2 genetic fragment for PCR, and F2 and R2 are used to expand CMV-ORF2-polyA genetic fragment, F3 and R3 is for expanding TKU-CMV-ORF2-polyA-TKL genetic fragment；

(1.2), the synthesis of 2 gene of PCV2 genome ORF and pZJ-1 carrier

It is respectively synthesized ORF2 gene and pZJ-1 carrier, ORF2 gene order is as shown in SEQ ID No.7, pZJ-1 carrier sequence As shown in SEQ ID No.8；

(1.3), construction recombination plasmid pZJ-ORF2

Distinguish double digestion ORF2 genetic fragment and pZJ-1 carrier using Kpn I, Hind III, ORF2 after recycling digestion and PZJ-1 passes through T₄DNA ligase is attached, and carries out plasmid conversion later, bacterium solution PCR screening positive clone will be with positive gram Grand opposite bacterium solution expands culture, and extracts plasmid, which is named as pZJ-ORF2；

(1.4), Nhe I and Spe I double digestion recombinant plasmid pZJ-ORF2

Using the DNA of recombinant plasmid pZJ-ORF2 as template, using F2 and R2 as primer, pcr amplification reaction is carried out, after the completion of amplification, Agarose gel electrophoresis simultaneously recycles pcr amplification product；Nhe I and Spe I double digestion pcr amplification product recycles digestion products, obtains Obtain CMV-ORF2-polyA genetic fragment；

(1.5), Spe I single endonuclease digestion pTK carrier and dephosphorylation process

Using Spe I single endonuclease digestion pTK carrier, dephosphorylation, purifies again after purification；

(1.6), CMV-ORF2-polyA genetic fragment and the processed pTK carrier digestion products of step (1.5) pass through T₄DNA connects It connects enzyme to be attached, carries out plasmid conversion later, bacterium solution PCR screening positive clone carries out the bacterium solution opposite with positive colony Expand culture, extract plasmid, which is named as pTK-ORF2；

(2), recombinant virus rPRV-gE^-/TK^--ORF2⁺Building

(2.1), using recombinant plasmid pTK-ORF2 as template, using F3 and R3 as primer, pcr amplification reaction, Ago-Gel are carried out Electrophoresis simultaneously recycles, and obtains TKU-CMV-ORF2-PloyA-TKL genetic fragment；

(2.2), PRV gene-deleted strain rPRV-gE is extracted^-/TK^-/EGFP⁺The genomic DNA of recombinant virus；

(2.3), by rPRV-gE^-/TK^-/EGFP⁺Genome and TKU-CMV-ORF2-PloyA-TKL genetic fragment cotransfection are extremely 293T cell, plaque purification obtain recombinant virus rPRV-gE^-/TK^-/ORF2⁺。

Preferably, in step (1.5), the pTK carrier is the plasmid pTK that applicant voluntarily constructs；In step (2.2), The PRV gene-deleted strain rPRV-gE^-/TK^-/EGFP⁺The recombination fluorescence virus HN-QYY-gE voluntarily constructed for applicant^-/TK^-/ EGFP⁺；Plasmid pTK and recombination fluorescence virus HN-QYY-gE^-/TK^-/EGFP⁺Construction step it is as follows:

S1, missing gE gene:

S1.1, by porcine pseudorabies virus variant HN-QYY(depositary institution: China Committee for Culture Collection of Microorganisms is general Logical microorganism center, preservation address: preservation date: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 is protected on December 22nd, 2017 Hiding number: CGMCC No.15192) it is seeded to the vero cell for covering with single layer, when 80% or more lesion occurs in cell, receive Virus liquid, multigelation are obtained, centrifugation takes supernatant, and phenol chloroform method extracts variant HN-QYY virus genom DNA, saves standby With；

S1.2, using variant HN-QYY virus genom DNA as template, utilize primer gEL-f/gEL-r and gER-f/gER-r point Other PCR amplification gE gene upstream and downstream homology arm gEL and gER, purified pcr product after agarose gel electrophoresis identification, measurement DNA contain Amount, saves backup；

Wherein, primer sequence is respectively as follows:

GEL-f: as shown in SEQ ID No.9；GEL-r: as shown in SEQ ID No.10；

GER-f: as shown in SEQ ID No.11；GER-r: as shown in SEQ ID No.12；

S1.3, EcoR I and the bis- enzymes of Spe I double digestion gEL, Spe I and Hind III double digestion gER, EcoR I and Hind III PMD18-T carrier is cut, gEL, gER, pMD18-T after recycling digestion；

S1.4, gEL after the recovery and gER are connected on pMD18-T after the recovery, carry out plasmid conversion, bacterium solution PCR sieve later Positive colony is selected, the bacterium solution opposite with positive colony is expanded culture, extracts plasmid, which is named as pgE；

S1.5, Spe I single endonuclease digestion plasmid pgE, dephosphorylation, purifies again after purification；

S1.6, Nhe I and Spe I double digestion pEGFP-N1 carrier, the pEGFP-N1 after recycling digestion；By pEGFP- after the recovery N1 is connected on the pgE after dephosphorylation, carries out plasmid conversion later, bacterium solution PCR screening positive clone will be with positive colony phase Pair bacterium solution expand culture, extract plasmid, which is named as pgE-EGFP；

S1.7, building recombination fluorescence virus HN-QYY-gE^-/EGFP⁺

By variant HN-QYY virus genom DNA, pgE-EGFP cotransfection obtained by step S1.1, to 293T cell, plaque is pure Change, until green fluorescence occur in all cytopathies, obtains recombination fluorescence virus HN-QYY-gE^-/EGFP⁺；

S1.8, removal green fluorescence gene

By HN-QYY-gE^-/EGFP⁺It is Plaque-purified with pcGlobin2-Cre plasmid co-transfection to 293T cell, until all thin Born of the same parents' lesion without fluorescence, obtains porcine pseudorabies virus variant HN-QYY-gE^-(depositary institution: Chinese microorganism strain is protected Administration committee's common micro-organisms center is hidden, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date: On 01 02nd, 2018, deposit number: CGMCC No.15200)；

S2, missing TK gene:

S2.1, by variant HN-QYY-gE obtained by step S1.8^-It is seeded to the vero cell for covering with single layer, when cell occurs 80% When the above lesion, virus liquid, multigelation are harvested, centrifugation takes supernatant, and phenol chloroform method extracts variant HN-QYY-gE^- Virus genom DNA saves backup；

S2.2, with variant HN-QYY-gE^-Virus genom DNA is template, utilizes primer TKL-f/TKL-r and TKR-f/ TKR-r distinguishes PCR amplification TK gene upstream and downstream homology arm TKL and TKR, and purified pcr product after agarose gel electrophoresis identification is surveyed Determine DNA content, saves backup；

Wherein, primer sequence is respectively as follows:

TKL-f: as shown in SEQ ID No.13；TKL-r: as shown in SEQ ID No.14；

TKR-f: as shown in SEQ ID No.15；TKR-r: as shown in SEQ ID No.16；

S2.3, EcoR I and the bis- enzymes of Spe I double digestion TKL, Spe I and Hind III double digestion TKR, EcoR I and Hind III PMD18-T carrier is cut, TKL, TKR, pMD18-T after recycling digestion；

S2.4, TKL after the recovery and TKR are connected on pMD18-T after the recovery, carry out plasmid conversion, bacterium solution PCR sieve later Positive colony is selected, the bacterium solution opposite with positive colony is expanded culture, extracts plasmid, which is named as pTK；

S2.5, Spe I single endonuclease digestion plasmid pTK, dephosphorylation, purifies again after purification；

S2.6, Nhe I and Spe I double digestion pEGFP-N1 carrier, the pEGFP-N1 after recycling digestion；By pEGFP- after the recovery N1 is connected on the pTK after dephosphorylation, carries out plasmid conversion later, bacterium solution PCR screening positive clone will be with positive colony phase Pair bacterium solution expand culture, extract plasmid, which is named as pTK-EGFP；

S2.7, building recombination fluorescence virus HN-QYY-gE^-/TK^-/EGFP⁺

By variant HN-QYY-gE obtained by step S2.1^-Virus genom DNA, pTK-EGFP cotransfection to 293T cell, plaque Purifying obtains recombination fluorescence virus HN-QYY-gE until green fluorescence occur in all cytopathies^-/TK^-/EGFP⁺。

Beneficial effects of the present invention: recombinant virus rPRV-gE of the present invention^-/TK^-/ORF2⁺Can inducing mouse generation be directed to The specific antibody of PCV2, anti-PRV neutralizing antibody titers can reach 1: 64；Illustrate that the recombinant virus has preferable immunogene Property, a kind of alternative vaccine strain is provided for two kinds of viral infection of PRV and PCV2 can be prevented simultaneously.

Detailed description of the invention

Fig. 1: porcine pseudorabies virus variant HN-QYY gE gene PCR identification, M:2000 DNA marker；1:HN- The PCR amplification of QYY gE gene；2:vero cell controls.

The PCR identification of Fig. 2: pgE and pgE-EGFP plasmid, M:15000bp DNA marker；The PCR amplification of 1:pgE；2: The PCR amplification of pgE-EGFP.

Fig. 3: porcine pseudorabies virus gE gene-deleted strain HN-QYY-gE^-PCR identification, M:250bp DNA marker, 1: GE-f/gE-r is to HN-QYY-gE^- The PCR amplification of gE gene, 2:gEL-f/gER-r is to HN-QYY-gE^-PCR amplification；3: GEL-f/gER-r is to HN-QYY-gE^-/EGFP⁺PCR amplification.

Fig. 4: pTK plasmid and HN-QYY-gE^-/TK^-/EGFP⁺The PCR identification of recombinant virus, M:15000bp DNA marker；The PCR amplification of 1:pTK；2:HN-QYY-gE^-/TK^-/EGFP⁺PCR amplification.

Fig. 5: HN-QYY-gE^-/TK^-The PCR identification of recombinant virus, M:250bp DNA marker；1:TK-f/TK-r pairs HN-QYY-gE^-The PCR amplification of TK gene；2:TK-f/TK-r is to HN-QYY-gE^-/TK^-The PCR amplification of TK gene.

Fig. 6: recombinant plasmid pZJ-ORF2 digestion identification, M:250bp Ladder, 1:Kpn I single endonuclease digestion recombinant plasmid PZJ-ORF2,2:Kpn I, Hind III double digestion recombinant plasmid pZJ-ORF2.

Fig. 7: Nhe I and Spe I double digestion recombinant plasmid pZJ-ORF2, M:250bp Ladder, 1-3:Nhe I and Spe I double digestion recombinant plasmid pZJ-ORF2.

Fig. 8: recombinant plasmid pTK-ORF2, M:DL250 ladder, 1-2:EcoR I, Hind III double digestion are identified in digestion Recombinant vector pTK-ORF2.

Fig. 9: the Western blot detection of Cap protein expression, M: pre-dyed protein molecular quality standard, 1: blank control, 2: negative control, 3: recombinant virus rPRV-gE^-/TK^-/ORF2⁺Albumen after inoculation BHK-21 cell.

Figure 10: PCV2 antibody level changes line chart.

Figure 11: each anti-PRV neutralizing antibody of immune group mouse is horizontal.

Specific embodiment

The separation of embodiment 1-- porcine pseudorabies virus variant HN-QYY is identified

Applicant shreds, in the brain tissue from Luoyang City pig farm acquisition morbid pig in 2012 by mass/volume ratio g/ Sterilizing PBS dissolution is added in mL 1: 5, is smashed with tissue refiner, -70 DEG C, room temperature multigelation 3 times, 8000rpm centrifugation 10min；Supernatant is taken, with 0.22 μm of disposable filter filtration sterilization, resulting tissue fluid is inoculated into covers with list at 1: 5 by volume On the vero cell of layer, virus liquid, -70 DEG C, room temperature multigelation 3 times, by virus liquid are harvested when lesion reaches 80% 8000rpm is centrifuged 10min, takes supernatant, and the vero cell of single layer is covered in renewed vaccination, and 5 generation of blind passage is (from there is the generation of lesion Rise), after lesion is stablized, harvest virus is Plaque-purified to get porcine pseudorabies virus variant HN-QYY simultaneously -70 DEG C of preservations.

Porcine pseudorabies virus variant HN-QYY virus genom DNA, specific steps are extracted with phenol chloroform method are as follows:

(1), -70 DEG C of virus liquid, room temperature multigelation after will be Plaque-purified 3 times；

(2), 350 μ L of virus liquid supernatant is taken to be added to 1.5mL centrifuge tube, in 350 μ L RIPA lysate of Guan Zhongjia and 10 μ L albumen Enzyme K is in 56 DEG C of 2 h of water-bath；

(3), the Tris saturated phenol of 700 μ L is added, mixes 3min, then 12000rpm is centrifuged 10min in 4 DEG C of centrifuges；

(4), it draws supernatant liquid and new centrifuge tube is added, add the mixed of the Tris saturated phenol of 700 μ L, chloroform and isoamyl alcohol Conjunction liquid (Tris saturated phenol: chloroform: isoamyl alcohol=25: 24: 1, volume ratio), 3min is mixed, 12000rpm centrifugation in 4 DEG C of centrifuges 10min；

(5), Aspirate supernatant is added to new centrifuge tube, and the mixed liquor (chloroform: isoamyl alcohol of isometric chloroform and isoamyl alcohol is added =24: 1, volume ratio), 3min is mixed, 12000rpm is centrifuged 10min in 4 DEG C of centrifuges；

(6), new centrifuge tube is added in Aspirate supernatant, is added isometric chloroform, mixes 3min, in 4 DEG C of centrifuges 12000rpm is centrifuged 10min；

(7), new centrifuge tube is added in 400 μ L of Aspirate supernatant, and the anhydrous of -20 DEG C of process freezings of 2.5 volumes times is then added Ethyl alcohol, -20 DEG C of precipitating 2h；

(8), sample is taken out, 12000rpm is centrifuged 20min in 4 DEG C of centrifuges, abandons supernatant, stays white precipitate, and 1000 μ L is added to pass through Cross the 75v% ethyl alcohol of -20 DEG C of freezings, mixings of turning upside down, 12000rpm centrifugation 10min in 4 DEG C of centrifuges；

(9), alcohol is discarded, static 3min, is finished to alcohol volatilization, the TE solution of 30 μ L, 4 DEG C of dissolution 30min is added DNA is obtained, -20 DEG C save backup.

The identification of porcine pseudorabies virus variant HN-QYY uses gE gene PCR method, and primer is as shown in table 1.GE base Because of PCR reaction system are as follows: 1 μ L, Taq archaeal dna polymerase (5 U/ μ L) 0.5 of 2xGC Buffer 25 μ L, dNTP (10 mmol/ μ L) μ L, upstream and downstream primer (5 OD) each 1 μ L, DNA profiling (100 μ g/mL) 4 μ L, sterilizing distilled water complement to 50 μ L；React item Part: 95 DEG C of 5 min of initial denaturation；95 DEG C of 30 s of denaturation, 60 DEG C of annealing 30s, 72 DEG C of extension 2min, 30 recycle；Last 72 DEG C 10min；PCR product is detected with agarose gel electrophoresis, the result is shown in Figure 1, shows that separated strain is porcine pseudorabies Virus variant.

Embodiment 2-- porcine pseudorabies virus variant HN-QYY-gE^-/TK^-/EGFP⁺And HN-QYY-gE^-/TK^-Structure Construction method

Construction step is as follows:

S1, missing gE gene:

After vero cell covers with single layer in S1.1, T25 cell bottle, nutrient solution is discarded, is washed 2 times with sterile PBS, 30 μ L are inoculated with Deposit number is the variant HN-QYY, 37 DEG C of CO of CGMCC No.15192₂It is incubated for 1h in incubator, discards virus liquid, is added The maintaining liquid (DMEM containing 2% fetal calf serum) of 6mL, continues 37 DEG C of CO₂It is cultivated in incubator, when 80% or more disease occurs in cell When change, virus liquid, -70 DEG C, room temperature multigelation 3 times are harvested, 8000rpm is centrifuged 10min, takes supernatant, phenol chloroform method Variant HN-QYY virus genom DNA is extracted, -20 DEG C save backup；

S1.2, using variant HN-QYY virus genom DNA as template, utilize primer gEL-f/gEL-r and gER-f/gER-r point Other PCR amplification gE gene upstream and downstream homology arm gEL and gER, purified pcr product after agarose gel electrophoresis identification, measurement DNA contain Amount, -20 DEG C save backup；

Wherein, primer sequence is as shown in table 2:

Pcr amplification reaction system are as follows: 25 μ L, dNTP(10 mmol/ μ L of 2xGC Buffer) 1 μ L, Taq archaeal dna polymerase (5 U/ μ L) 0.5 μ L, upstream and downstream primer (5 OD) each 1 μ L, DNA profiling (100 μ g/mL) 4 μ L, moisturizing to 50 μ L；PCR reaction Condition are as follows: 95 DEG C of 5 min of initial denaturation；95 DEG C of 30 s of denaturation, 60 DEG C of annealing 30s, 72 DEG C of extension 80s, 30 recycle；Finally 72 DEG C of 10 min of extension；

S1.3, EcoR I and the bis- enzymes of Spe I double digestion gEL, Spe I and Hind III double digestion gER, EcoR I and Hind III PMD18-T carrier is cut, gEL, gER, pMD18-T after recycling digestion, -20 DEG C save backup；；

Wherein, digestion system is respectively as follows: EcoR I(15 U/ μ L) 1.5 μ L, Spe I(15 U/ μ L) 1.5 μ L, segment gEL 35 μ L of μ g, 10 × buffer, moisturizing to 50 μ L；Spe I(15 U/ μ L) 1.5 μ L, Hind III(15 U/ μ L) 1.5 μ 35 μ L of μ g, 10 × buffer of L, segment gER, moisturizing to 50 μ L；EcoR I(15 U/ μ L) 1 μ L, Hind III(15 U/ μ L) 1 μ L, pMD18-T carrier, 25 μ L of μ g, 10 × buffer, moisturizing to 50 μ L；Digestion condition is equal are as follows: 37 DEG C of water-baths 4 h of middle incubation；

S1.4, gEL after the recovery and gER are connected on pMD18-T after the recovery, carry out plasmid conversion, bacterium solution PCR sieve later Positive colony is selected, the bacterium solution opposite with positive colony is expanded culture, extracts plasmid, which is named as pgE, and -20 DEG C It saves backup；

Wherein, linked system are as follows: T₄DNA ligase (350 U/ μ L) 1 μ L, 10 × T₄1 μ L of Ligase buffer, gene Segment and carrier segments (with molar ratio computing, gEL: gER: pMD18-T=5: 5: 1), and moisturizing to 10 μ L；Condition of contact are as follows: 16 DEG C Connect 16h；

The specific steps of plasmid conversion are as follows: 100 μ L of competent escherichia coli cell DH5 α is taken out, is placed in mixture of ice and water, it will 10 μ L connection products are all added in competent cell, and gently concussion mixes, and stand 30 min；Competent cell is placed in later 42 DEG C, heat shock 90s, then be placed in mixture of ice and water and stand 5 min, the addition not resistant LB culture medium of 1mL, 37 DEG C, 200rpm shaking table culture 1h, is then centrifuged for, and stays 200 μ L supernatants, is uniformly mixed, takes the bacterium solution of 100 μ L supernatants to be evenly coated in and contain Have on the solid LB plate (100 μ g/mL of ampicillin content, similarly hereinafter) of ampicillin (Amp+) resistance, 37 DEG C of constant temperature trainings 16 h are supported, picking colony to 1mL contains in the LB culture medium of Amp+ resistance, and 37 DEG C, 200rpm shaking table culture 12h are prepared into bacterium Liquid carries out bacterium solution PCR；

Bacterium solution PCR amplification system: 25 μ L, dNTP(10 mmol/ μ L of 2xGC Buffer) 1 μ L, Taq archaeal dna polymerase (5 U/ μ L) 0.5 μ L, primer gEL-f and gER-r(5 OD) each 1 μ L, 4 μ L of bacterium solution, moisturizing to 50 μ L；PCR reaction condition are as follows: 95 DEG C pre- It is denaturalized 5 min；95 DEG C of 30 s of denaturation, 60 DEG C of annealing 30s, 72 DEG C of 150 s of extension, 30 recycle；Last 72 DEG C extend 10 min；Identification (see figure 2) is carried out with agarose gel electrophoresis later, picking electrophoretic band position is the positive in the bacterium solution of 2600 bp Bacterium solution；

Expand incubation step are as follows: be connected to positive bacterium solution in 15 mL LB culture mediums at 1: 100 by volume, 37 DEG C of shaking tables 200 R/min shakes 14 h of bacterium；

S1.5, Spe I single endonuclease digestion plasmid pgE, dephosphorylation after purification, then purified with PCR product kit, -20 DEG C of guarantors It deposits spare；

Wherein, digestion system are as follows: Spe I(15 U/ μ L) 2 μ L, 45 μ L of μ g, 10 × buffer of plasmid pgE, moisturizing to 50 μ L；Digestion condition are as follows: be incubated for 3h in 37 DEG C of water-baths；

Dephosphorylation system are as follows: dephosphorylation enzyme (1 U/ μ L) 2 μ L, the 2 μ g, 10 × buffer 5 of plasmid pgE after digestion μ L, moisturizing to 50 μ L；Dephosphorylation condition are as follows: be incubated for 3h in 37 DEG C of water-baths；

S1.6, Nhe I and Spe I double digestion pEGFP-N1 carrier (being purchased from Hunan Feng Hui Biotechnology Co., Ltd), recycle enzyme EGFP-N1 segment after cutting；On pgE after EGFP-N1 segment after the recovery to be connected to dephosphorylation, plasmid is carried out later and is turned Change, bacterium solution PCR screening positive clone expands culture the bacterium solution opposite with positive colony, extracts plasmid, plasmid name For pgE-EGFP；

Wherein, digestion system are as follows: Nhe I(15 U/ μ L) 1 μ L, Spe I(15 U/ μ L) 1 μ L, pEGFP-N1 2 μ g, 10 × 5 μ L of buffer, moisturizing to 50 μ L；Digestion condition are as follows: be incubated for 3h in 37 DEG C of water-baths；

Linked system are as follows: T₄DNA ligase (350 U/ μ L) 1 μ L, 10 × T₄1 μ L of Ligase buffer, genetic fragment With carrier segments (with molar ratio computing, EGFP-N1: pgE=5: 1), and moisturizing to 10 μ L；Condition of contact are as follows: 16 DEG C of connection 16h；

Plasmid conversion, bacterium solution PCR and the step of expand culture with step S1.4, only bacterium solution PCR is with agarose gel electrophoresis After carrying out identification (see figure 2), picking electrophoretic band position is positive bacterium solution in the bacterium solution of 4500bp or so；

S1.7, building recombination fluorescence virus HN-QYY-gE^-/EGFP⁺

Using liposome LipofectamineTM2000 by HN-QYY viral genome, pgE-EGFP according to g: 3 μ g of 1 μ ratio It being transfected: the 293T cell for growing to 80% in six orifice plates being discarded into nutrient solution first, the serum-free DMEM of 1mL is added in every hole, 37℃ CO₂1h is incubated in incubator；Two centrifuge tubes are taken, every plus 300 μ L serum-free DMEM without double antibody, then the first pipe adds 1 μ g of variant HN-QYY virus genom DNA obtained by step S1.1,3 μ L of liposome, the second pipe plus 3 μ g of plasmid pgE-EGFP, 9 μ L of liposome, all concussion mixes for the first pipe and the second pipe, stands 5min, and two pipes are merged into a pipe later, and piping and druming mixes, Static 30min is added in 293T cell, 37 DEG C of CO₂It is incubated for 6 h in incubator, discards culture solution, changes into containing 2% tire ox blood Clear DMEM, 37 DEG C of CO₂Continue to cultivate in incubator, be harvested after 36h, -70 DEG C, after room temperature multigelation, single layer is covered in inoculation Vero cell, when cell occur 80% lesion when receive poison；

It is Plaque-purified: take virus liquid, be centrifuged, take supernatant supernatant is inoculated in by 10 doubling dilutions grow to single layer vero it is thin Six orifice plate of born of the same parents, every 1000 μ L of hole discard virus liquid after adsorbing 1h, and 2% agarose is added and 2 × DMEM(contains 2% fetal calf serum) with 2 mL of mixture that volume ratio 1: 1 forms, when cytopathy to appear, with glimmering in picking highest dilution under fluorescence microscope The plaque of light, and six orifice plate of vero cell for growing to single layer is inoculated in again according to 10 times of doubling dilutions, occur to cell green When color fluorescence lesion, selects the maximum hole picking of dilution to continue to be inoculated with vero cell with the plaque of green fluorescence, so purify There is the lesion of green fluorescence to all plaques, carries out Ago-Gel after PCR amplification to it using primer gEL-f/gER-r Electroresis appraisal (amplification system and the rapid S1.4 bacterium solution PCR amplification system of conditional synchronization and condition, see Fig. 3) saves this generation virus, It is named as HN-QYY-gE^-/EGFP⁺；

Amplification virus on vero cells, specific steps are as follows: when vero cell grows to single layer in T25 cell bottle, discard old The virus liquid of 20 μ L is diluted with the DMEM 2mL of serum-free, is added in cell bottle, 37 DEG C of CO by culture solution₂It is incubated in incubator 1h is educated, changes the DMEM containing 2% fetal calf serum, 37 DEG C of CO into₂Continue to cultivate in incubator, when cytopathy 80% after harvest, -70 DEG C, room temperature multigelation, take appropriate virus liquid, phenol chloroform DNA measures content, and -20 DEG C save backup；

S1.8, removal green fluorescence gene

By HN-QYY-gE^-/EGFP⁺With pcGlobin2-Cre plasmid according to g: 3 μ g of 1 μ ratio according to step S1.7 method It transfects to 80% 293T cell, harvests after 36h, after -70 DEG C, room temperature multigelation 3 times, the vero cell of single layer is covered in inoculation, Virus liquid is harvested when 80% lesion occurs in cell；According to the Plaque-purified virus of the method for step S1.7, until all cells Lesion carries out agarose gel electrophoresis identification (amplification body after PCR amplification using primer gEL-f/gER-r without fluorescence to it System and the rapid S1.4 bacterium solution PCR amplification system of conditional synchronization and condition, are shown in Fig. 3), this generation virus is saved, pseudorabies are named as Sick virus variant HN-QYY-gE^-；

Porcine pseudorabies virus variant HN-QYY-gE is extracted with phenol chloroform method^-Virus genom DNA, using primer gE-f and (amplification system and condition are the same as HN-QYY in embodiment 1 for agarose gel electrophoresis identification after gE-r carries out PCR amplification to its gE gene GE gene PCR amplification system and condition, see Fig. 3), the results showed that HN-QYY-gE^-GE gene is successfully lacked；

S2, missing TK gene:

After vero cell covers with single layer in S2.1, T25 cell bottle, nutrient solution is discarded, is washed 2 times with sterile PBS, 30 μ L are inoculated with Variant HN-QYY-gE obtained by step S1.8^-, 37 DEG C of CO₂It is incubated for 1h in incubator, discards virus liquid, the maintaining liquid of 6mL is added (DMEM containing 2% fetal calf serum) continues 37 DEG C of CO₂It cultivates in incubator, when 80% or more lesion occurs in cell, receives Virus liquid, -70 DEG C, room temperature multigelation 3 times are obtained, 8000rpm is centrifuged 10min, takes supernatant, and phenol chloroform method extracts variant HN-QYY-gE^-Virus genom DNA, -20 DEG C save backup；

S2.2, with variant HN-QYY-gE^-Virus genom DNA is template, utilizes primer TKL-f/TKL-r and TKR-f/ TKR-r distinguishes PCR amplification TK gene upstream and downstream homology arm TKL and TKR, and purified pcr product after agarose gel electrophoresis identification is surveyed Determine DNA content, saves backup；

Wherein, primer sequence is as shown in table 3:

Pcr amplification reaction system and the rapid S1.2 of conditional synchronization；

S2.3, EcoR I and the bis- enzymes of Spe I double digestion TKL, Spe I and Hind III double digestion TKR, EcoR I and Hind III PMD18-T carrier is cut, TKL, TKR, pMD18-T after recycling digestion, -20 DEG C save backup；；

Wherein, digestion system and the rapid S1.3 of conditional synchronization；

S2.4, TKL after the recovery and TKR are connected on pMD18-T after the recovery, carry out plasmid conversion, bacterium solution PCR sieve later Positive colony is selected, the bacterium solution opposite with positive colony is expanded culture, extracts plasmid, which is named as pTK, and -20 DEG C It saves backup；

Wherein, linked system and condition, plasmid step of converting, bacterium solution PCR amplification system and condition, the expansion same step of incubation step S1.4, only primer when bacterium solution PCR is TKL-f and TKR-r, and agarose gel electrophoresis qualification result is shown in Fig. 4；

S2.5, Spe I single endonuclease digestion plasmid pTK, dephosphorylation after purification, then purified with PCR product kit, -20 DEG C of guarantors It deposits spare；

Wherein, digestion system and condition, dephosphorylation system and the rapid S1.5 of conditional synchronization；

S2.6, Nhe I and Spe I double digestion pEGFP-N1 carrier (being purchased from Hunan Feng Hui Biotechnology Co., Ltd), recycle enzyme EGFP-N1 segment after cutting；On pTK after EGFP-N1 segment after the recovery to be connected to dephosphorylation, plasmid is carried out later and is turned Change, bacterium solution PCR screening positive clone expands culture the bacterium solution opposite with positive colony, extracts plasmid, plasmid name For pTK-EGFP；

Wherein, linked system and condition, plasmid step of converting, bacterium solution PCR amplification system and condition, the expansion same step of incubation step S1.4；

S2.7, building recombination fluorescence virus HN-QYY-gE^-/TK^-/EGFP⁺

It is transfected using liposome LipofectamineTM2000: first being abandoned the 293T cell for growing to 80% in six orifice plates Remove nutrient solution, the serum-free DMEM, 37 DEG C of CO of every hole addition 1mL₂1h is incubated in incubator；Two centrifuge tubes are taken, every adds 300 μ L serum-free DMEM without double antibody, then the first pipe adds variant HN-QYY-gE obtained by step S2.1^-Virus genom DNA 1 μ g, 3 μ L of liposome, the second pipe plus 3 μ g of plasmid pTK-EGFP, 9 μ L of liposome, all concussion mixes for the first pipe and the second pipe, quiet 5min is set, two pipes are merged into a pipe later, piping and druming mixes, and static 30min is added in 293T cell, 37 DEG C of CO₂Culture It is incubated for 6 h in case, discards culture solution, changes the DMEM containing 2% fetal calf serum, 37 DEG C of CO into₂Continue to cultivate in incubator, after 36h Harvest, -70 DEG C, after room temperature multigelation, the vero cell of single layer is covered in inoculation, receives poison when 80% lesion occurs in cell；

It is Plaque-purified: take virus liquid, be centrifuged, take supernatant supernatant is inoculated in by 10 doubling dilutions grow to single layer vero it is thin Six orifice plate of born of the same parents, every 1000 μ L of hole discard virus liquid after adsorbing 1h, and 2% agarose is added and 2 × DMEM(contains 2% fetal calf serum) with 2 mL of mixture of volume ratio 1: 1, when cytopathy to appear, with fluorescence in picking highest dilution under fluorescence microscope Plaque, and six orifice plate of vero cell for growing to single layer is inoculated in again according to 10 times of doubling dilutions, it is glimmering green occur to cell When light lesion, selects the maximum hole picking of dilution to continue to be inoculated with vero cell with the plaque of green fluorescence, be so purified to institute There is plaque the lesion of green fluorescence occur, carries out agarose gel electrophoresis after PCR amplification to it using primer TKL-f/TKR-r It identifies (amplification system and the rapid S1.4 bacterium solution PCR amplification system of conditional synchronization and condition, see Fig. 4), this generation virus is saved, name For HN-QYY-gE^-/TK^-/EGFP⁺；

Amplification virus on vero cells, specific steps are as follows: when vero cell grows to single layer in T25 cell bottle, discard old The virus liquid of 20 μ L is diluted with the DMEM 2mL of serum-free, is added in cell bottle, 37 DEG C of CO by culture solution₂It is incubated in incubator 1h is educated, changes the DMEM containing 2% fetal calf serum, 37 DEG C of CO into₂Continue to cultivate in incubator, when cytopathy 80% after harvest, -70 DEG C, room temperature multigelation, take appropriate virus liquid, phenol chloroform DNA measures content, and -20 DEG C save backup；

S2.8, removal green fluorescence gene

By HN-QYY-gE^-/TK^-/EGFP⁺With pcGlobin2-Cre plasmid according to g: 3 μ g of 1 μ ratio according to step S2.7 side Method is transfected to 80% 293T cell, is harvested after 36h, and after -70 DEG C, room temperature multigelation 3 times, the vero that single layer is covered in inoculation is thin Born of the same parents harvest virus liquid when 80% lesion occurs in cell；According to the Plaque-purified virus of the method for step S2.7, until all Cytopathy is without fluorescence, and agarose gel electrophoresis identification (is expanded after carrying out PCR amplification to it using primer TKL-f/TKR-r Increasing system and the rapid S1.4 bacterium solution PCR amplification system of conditional synchronization and condition), this generation virus is saved, porcine pseudorabies are named as Virus variant HN-QYY-gE^-/TK^-；

Porcine pseudorabies virus variant HN-QYY-gE is extracted with phenol chloroform method^-/TK^-Virus genom DNA.Utilize primer TK- F(such as SEQ ID No.19) and TK-r(such as SEQ ID No.20) respectively to HN-QYY-gE^-And HN-QYY-gE^-/TK^-In TK Gene carries out agarose gel electrophoresis identification (the gE gene of amplification system and condition with HN-QYY in embodiment 1 after PCR amplification PCR amplification system and condition, are shown in Fig. 5), the results showed that HN-QYY-gE^-/TK^-TK gene is successfully lacked.

Embodiment 3-- recombinant porcine pseudorabies Strain rPRV-gE^-/TK^-/ORF2⁺Construction method

Construction step is as follows:

(1), the building of recombinant plasmid pTK-ORF2

(1.1), the primer of PCR amplification ORF2, CMV-ORF2-poly A, TKU-CMV-ORF2-polyA-TKL, primer letter are designed Breath such as table 4:

Wherein: F1 and R1 for identifying ORF2 genetic fragment, F2 and R2 for expanding CMV-ORF2-polyA genetic fragment, F3 and R3 is for expanding TKU-CMV-ORF2-polyA-TKL genetic fragment；

(1.2), the synthesis of 2 gene of PCV2 genome ORF and pZJ-1 carrier

PCV2 is synthesized according to the gene order of the GenBank PCV2 JZ-8 strain (gene accession number are as follows: KU960934) announced ORF2 gene, and its 5 ' end addition Kpn I restriction enzyme site, 3 ' end addition Hind III digestion sites, ORF2 gene order by Sangon Biotech's synthesis, ORF2 gene order is as shown in SEQ ID No.7；

PZJ-1 carrier is synthesized by Sangon Biotech (Shanghai) Co., Ltd., pZJ-1 carrier sequence such as SEQ ID No.8 It is shown；

(1.3), construction recombination plasmid pZJ-ORF2

Double digestion ORF2 genetic fragment and pZJ-1 carrier are distinguished using Kpn I, Hind III, use SanPrep pillar PCR respectively ORF2 and pZJ-1 after Product Purification Kit purification and recovery digestion, pass through T₄DNA ligase is attached, and carries out matter later Grain conversion, bacterium solution PCR screening positive clone expands culture the bacterium solution opposite with positive colony, with SanPrep pillar matter The a small amount of extraction agent boxes of grain DNA extract plasmid, Kpn I, the identification of Hind III double digestion, (digestion identification system as shown in Figure 6 (20 μ L): 10 × M Buffer 2 μ L, Kpn I, Hind III 2 μ g of each 1 μ L, plasmid pZJ-ORF2 add distilled water to 20 μ L； Digestion condition are as follows: 37 DEG C of 3 h of effect；Theoretically double digestion goes out two target fragments of 3500bp, 709bp, and single endonuclease digestion answers digestion to go out The segment of 4209bp), identify that correct plasmid is named as pZJ-ORF2；

Wherein, digestion system (100 μ L): 10 × M Buffer 10 μ L, Kpn I, each 2 μ L, ORF2 genetic fragment of Hind III or Each 3 μ g of pZJ-1 carrier, adds distilled water to 100 μ L；Digestion condition are as follows: 37 DEG C of 3 h of effect are added Loading Buffer and terminate Reaction；

Linked system: 10 × T₄Ligase Buffer 1 μ L, T₄Ligase(350U/ μ L) 1 μ L, ORF2 genetic fragment with PZJ-1 carrier is added with the ratio that molar ratio is 3: 1, adds water to 10 μ L；Condition of contact condition are as follows: 16 DEG C overnight；

Plasmid step of converting are as follows: connection product converts bacillus coli DH 5 alpha competent cell, amoxicillin resistance LB plate (100 μ g/mL of ampicillin content, similarly hereinafter) carries out preliminary screening, and picking individually clones addition and contains amicillin resistance 37 DEG C of 200 r/min of shaking table of LB culture medium shake 6 h of bacterium, carry out bacterium solution PCR；

Bacterium solution PCR amplification system: 2 × Es Taq MasterMix 25 each 1 μ L of μ L, F1, R1,4 μ L of bacterium solution template add distilled water To 50 μ L；PCR amplification condition: 95 DEG C of 5min, 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s expand 30 circulations, and 72 DEG C are prolonged Stretch 10min；

Expand incubation step are as follows: be connected to positive colony opposite bacterium solution in 15 mL LB culture mediums at 1: 100 by volume, 37 DEG C 200 r/min of shaking table shakes 14 h of bacterium；

(1.4), Nhe I and Spe I double digestion recombinant plasmid pZJ-ORF2

Using the DNA of recombinant plasmid pZJ-ORF2 as template, using F2 and R2 as primer, pcr amplification reaction is carried out, after the completion of amplification, Agarose gel electrophoresis simultaneously recycles pcr amplification product；(see Fig. 7, digestion is big out for Nhe I and Spe I double digestion pcr amplification product Small is the CMV-ORF2-poly A target gene fragment of 1500bp or so and the genetic fragment of 2700bp), utilize SanPrep column Formula Plasmid DNA plastic recovery kit recycles the digestion products that size is 1500bp or so, obtains CMV-ORF2-polyA gene piece Section；

Wherein, pcr amplification reaction system: 5 × PrimeSTARGXL Buffer, 10 μ L, 2.5mM dNTP Mixture, 4 μ L, Primers F 2 and each 1 μ L, PrimeSTARGXL DNA Polymerase(1.25 U/ μ L of R2) 1 μ L, 4 μ L of DNA profiling adds distilled water To 50 μ L；Amplification reaction condition: 98 DEG C of 15s, 60 DEG C of 10s, 68 DEG C of 40s expand 30 circulations, 68 DEG C of extension 10min；

Digestion system (100 μ L): 10 × M Buffer 10 μ L, Nhe I, Spe I each 2 μ L, recombinant plasmid pZJ-ORF2 PCR expansion Increase production 4 μ g of object, adds distilled water to 100 μ L；Digestion condition are as follows: 37 DEG C of 4 h of effect；

(1.5), Spe I single endonuclease digestion pTK carrier and dephosphorylation process

Using Spe I single endonuclease digestion pTK carrier (the plasmid pTK constructed for embodiment 2), digestion products are through SanPrep pillar PCR Product Purification Kit is recycled, and recovery product is carried out dephosphorylation, and dephosphorylation product carries out phenol chloroform, is taken out Mentioning product recycles SanPrep pillar PCR product purification kit to be purified；

Wherein, 2 μ L, pTK carrier of digestion system (100 μ L): 10 × M Buffer 10 μ L, Spe I, 4 μ g, adds distilled water extremely 100μL；Digestion condition are as follows: 37 DEG C of 4 h of effect；

Dephosphorylation system (50 μ L): Alkaline Phosphatase (Calf Intestine) (1 U/ μ L) 2 μ L, 10 × 5 μ L of Alkaline Phosphatase Buffer, the 2 μ g of pTK carrier after digestion, moisturizing to 50 μ L；Dephosphorylation condition Are as follows: 37 DEG C of 1 h of reaction；

Phenol chloroform step: dephosphorylation product is supplied into 100 μ L with Elution Buffer, Tris- balance phenol 100 is added μ stands 5min after mixing, 4 DEG C of 10000r/min are centrifuged 5min；It takes and stands 5min after being centrifuged after the imitative 100 μ L mixing of supernatant chlorination, 4 DEG C 10000r/min is centrifuged 5min；Supernatant is taken, adds appropriate Elution Buffer to supply 100 μ L, -20 DEG C save backup；

(1.6), CMV-ORF2-polyA genetic fragment and the processed pTK carrier digestion products of step (1.5) pass through T₄DNA connects It connects enzyme to be attached, carries out plasmid conversion later, bacterium solution PCR screening positive clone carries out the bacterium solution opposite with positive colony Expanding culture, extracts plasmid, EcoR I, Hind III double digestion are identified, as shown in Figure 8 (digestion identification system (20 μ L): 10 × M Buffer 2 μ L, EcoR I, Hind III 2 μ g of each 1 μ L, plasmid pTK-ORF2, add distilled water to 20 μ L；Digestion condition are as follows: 37 DEG C of 3 h of effect；Tetra- genetic fragments of 2700bp, 2400bp, 1700bp, 400bp should be theoretically obtained, it is shown in Fig. 8 and theoretical It is consistent, shows that pTK-ORF2 is constructed successfully), identify that correct plasmid is named as pTK-ORF2；

Wherein, linked system: 10 × T₄Ligase Buffer 1 μ L, T₄Ligase(350U/ μ L) 1 μ L, CMV-ORF2- PolyA genetic fragment and the processed pTK carrier digestion products of step (1.5) are added with the ratio that molar ratio is 3: 1, are added water to 10 μL；Condition of contact condition are as follows: 16 DEG C overnight；

Plasmid conversion, bacterium solution PCR system and condition expand the same step of incubation step (1.3), only drawing in bacterium solution PCR system Object is F2, R2；

(2), recombinant virus rPRV-gE^-/TK^--ORF2⁺Building

(2.1), using recombinant plasmid pTK-ORF2 as template, using F3 and R3 as primer, pcr amplification reaction, Ago-Gel are carried out Electrophoresis simultaneously recycles, and obtains TKU-CMV-ORF2-PloyA-TKL genetic fragment；

Wherein, pcr amplification reaction system: 5 × PrimeSTARGXL Buffer, 10 μ L, 2.5mM dNTP Mixture, 4 μ L, Each 1 μ L, PrimeSTARGXL DNA Polymerase(1.25 U/ μ L of upstream and downstream primer) 1 μ L, 4 μ L of DNA profiling adds distilled water To 50 μ L；Amplification reaction condition: 98 DEG C of 10s, 60 DEG C of 5s, 68 DEG C of 4min expand 30 circulations, 68 DEG C of extension 10min；

(2.2), the PRV gene-deleted strain HN-QYY-gE that phenol chloroform method extracting embodiment 2 obtains^-/TK^-/EGFP⁺Genomic DNA；

(2.3), cotransfection and plaque purification obtain recombinant virus rPRV-gE^-/TK^-/ORF2⁺:

(2.3.1), cultivate 293T cell 12 ~ for 24 hours in 6 porocyte culture plates, cell monolayer it is long to 80% or so for transfecting；

(2.3.2), 2 sterile 1.5mL EP pipes are taken, every pipe is separately added into the DMEM of 200 μ L, and TKU-CMV- is added in the first pipe 4 μ g of ORF2-PloyA-TKL genetic fragment, 12 μ L of transfection reagent Lipofectamine2000；HN-QYY- is added in second pipe gE^-/TK^-/EGFP⁺2 μ g of genome, 6 μ L of transfection reagent Lipofectamine2000；First pipe and the second pipe all shake It mixes, is stored at room temperature 5min, two pipes are merged into a pipe later, piping and druming mixes, and is stored at room temperature 30min；

(2.3.3), cell culture medium old in 6 porocyte culture plates is discarded, it is primary to wash cell with DMEM, then every hole is added Then (2.3.2) transfection mixture is added in 6 porocyte culture plates, while doing control of the hole without transfection by 500 μ L DMEM, Set 37 DEG C of 5% CO₂Incubator culture；

After (2.3.4), 6h, discards liquid in hole and add the DMEM for containing 2% fetal calf serum containing 2mL, be placed in 37 DEG C of 5% CO₂Culture Case continues to cultivate；

(2.3.5), to cytopathy to 80% or so, collects transfection cocktail and be inoculated with transfection cocktail centrifuging and taking supernatant Vero cell, absorption 1.5h discard transfection cocktail, and 2% agarose is added and 2 × DMEM(contains 2% fetal calf serum) with volume ratio 1: 12 mL of mixture；

It when (2.3.6), cytopathy to appear, is observed under fluorescence inverted microscope, picking unstressed configuration plaque simultaneously carries out plaque It is purified to 100% unstressed configuration, obtains recombinant virus rPRV-gE^-/TK^-/ORF2⁺。

Embodiment 4-- recombinant virus rPRV-gE^-/TK^-/ORF2⁺The expression of Cap protein

(1) by recombinant virus rPRV-gE^-/TK^-/ORF2⁺The long BHK-21 cell to single layer of inoculating cell, and be arranged and do not connect poison carefully Born of the same parents' blank control and rPRV-gE^-/TK^-/EGFP⁺(for the HN-QYY-gE in embodiment 2^-/TK^-/EGFP⁺) virus connect it is malicious negative right According to；37℃ 5% CO₂Incubator culture；

(2) it when BHK-21 cytopathy 80% or so, inhales the liquid abandoned in hole and gently to clean cell with PBS primary, with 100 μ L RIPA fine melt liquid (added with the protease inhibitors of 1mM) blows down cell, and obtained cell suspension is put in centrifuge tube, on ice 20min or so is cracked, every 5min is vortexed once, is put in -70 DEG C；Poison cell blank control and rPRV-gE are not met^-/TK^-/EGFP⁺ It connects malicious negative control and collects albumen according to same operation；

(3) each protein sample takes 20 μ L, boils 5min, 10000r/min centrifugation after 5 × loading buffer is added 1min；Pre-prepared SDS-PAGE protein adhesive is taken out, the protein sample that 20 μ L are handled well is added in each swimming lane, by electrophoresis apparatus Voltage is adjusted to 80V, closes power supply after bromjophenol blue all runs out of glue；

(4) transferring film: appropriately sized nitrocellulose filter (NC film), filter paper and separation gel are soaked in electricity and turned in liquid in advance；It will Film is placed on filter paper, then separation blob of viscose is gently placed on film, and driven out of bubble with glass bar, finally by the last layer filter paper It is covered on separation blob of viscose, drives bubble out of, make four tightly to fit together, press upper two lateral electrode, connect power supply, current stabilization 200mAh electricity turns 30min；

(5) it closes: after electricity turns, being taken out after NC film is rinsed 3 times in TBST buffer, then be soaked in containing 5%(g/ ML, similarly hereinafter) in the TBST confining liquid of defatted milk, 2h is first closed at room temperature, then is put in 4 DEG C overnight；

(6) primary antibody is incubated for: being washed film 3 times after closing with TBST, 5 min, is then added the dilution of volume ratio 1: 2000 and (uses every time Containing 5% defatted milk TBST confining liquid dilution) anti-Cap protein monoclonal antibody, in 60 r/min of shaking table react 2 h；

(7) secondary antibody is incubated for: being washed film 3 times, each 5min with TBST, the dilution of volume ratio 1: 5000 is then added (with containing 5% defatted milk TBST confining liquid dilution) HRP- rabbit-anti mouse secondary antibody, in shaking table 60 r/min be incubated for 1 h；

(8) develop the color: TBST is washed film 3 times, each 5min, is blotted the extra moisture of film surface only, in darkroom with clean filter paper In, ECL developing solution is uniformly dripped on film, develop the color 3min, observes result.

The Western-blot testing result of Cap protein is shown in Fig. 9, recombinant virus rPRV-gE^-/TK^-/ORF2⁺It is inoculated with BHK- There is a band in 27.8KD after 21 cells, is consistent with Cap protein theory size, illustrates recombinant virus rPRV-gE^-/TK^-/ ORF2⁺Cap protein can be expressed.

Embodiment 5-- recombinant virus rPRV-gE^-/TK^-/ORF2⁺Evaluation of Immunogenicity

1 material and method

1.1 experimental animal

6-8 week old female SPF kunming mice 20, it is purchased from experimental animal center of henan province.

1.2 vaccines and seed culture of viruses

Pig circular ring virus subunit vaccine (Ingelvac CircoFLEX) is purchased from the limited public affairs of Boehringer Ingelheim animal health Department, pseudo- mad (HB-98 plants) of disease live-vaccine of pig are purchased from Wuhan Ke Qian Biological Co., Ltd.；rPRV-gE^-/TK^-/ORF2⁺Recombination Virus is that the embodiment of the present invention 3 constructs.

1.3 immunization protocol

20 female KM mouse are randomly divided into 4 groups, every group 5.Recombinant virus rPRV-gE is immunized in A group^-/TK^-/ORF2⁺, B group Immune pig circular ring virus subunit vaccine, the pseudo- mad disease live-vaccine of C group immune swine, the immune DMEM culture solution of D group as negative control, Booster immunization is primary (immunizing dose and approach is same exempt from) after 4 weeks.Immune grouping situation is as shown in table 5.

1.4 serum collection

Docking blood sampling is carried out to every mouse in every group weekly, 37 DEG C of incubators of mouse blood are placed into 1h, then 4 DEG C are stayed overnight, 2500r/min is centrifuged 10min, separates serum.

1.5 indirect ELISAs detect anti-PCV2 antibody level in mice serum

(1) 96 hole elisa Plates are taken out from aluminium foil bag, every hole is coated with the PCV2 Cap protein of 0.25 μ g, is incubated in 37 DEG C of incubators 1 h, 4 DEG C overnight；

(2) every hole adds the TBST confining liquid containing 5% defatted milk of 200 μ L in ELISA Plate, and 37 DEG C of wet box are incubated for 1h, discard liquid in hole Body is patted dry on clean blotting paper, and PBST is washed 5 times, and each 5min pats dry ELISA Plate；

(3) by 1: 500 dilution by volume of the TBST confining liquid containing 5% defatted milk of each group mice serum to be checked, 100 holes μ L/ It being added in ELISA Plate, 37 DEG C of wet box are incubated for 2h, and liquid in hole is discarded, is patted dry on clean blotting paper, PBST washing 5 times, every time 5min pats dry ELISA Plate；

(4) by 1: 5000 dilution by volume of the TBST confining liquid containing 5% defatted milk of HRP sheep anti mouse secondary antibody, 100 holes μ L/ add Entering in ELISA Plate, 37 DEG C of wet box are incubated for 1h, and liquid in hole is discarded, is patted dry on clean blotting paper, PBST washing 5 times, every time 5min pats dry ELISA Plate；

(5) ELISA Plate, 100 holes μ L/ is added in one-component TMB developing solution, 37 DEG C of wet box are protected from light 10min；

(6) it is added 2mol/L concentrated sulfuric acid terminate liquid, 50 holes μ L/, in measuring every hole OD with microplate reader in 15min₄₅₀Value.

It is horizontal that 1.6 virus neutralization tests detect PRV neutralizing antibody in serum

(1) TCID of PRV QYY strain (for the HN-QYY in embodiment 1) is measured₅₀, and virus liquid is diluted to 200TCID₅₀/ 0.1mL；

(2) by the preparatory 56 DEG C of inactivations 30min of the mice serum of each group, 50 μ L are added in every hole in 96 porocyte culture plates DMEM, then the serum of equivalent is taken to be added in 96 orifice plates, it is 1: 2,1: 4,1: 8,1: 16,1 with 2 times of doubling dilution serum of DMEM: 32,1: 64,1: 128,1: 256, each dilution does six repetitions, and the virus liquid that equivalent has diluted is added in above-mentioned each hole, Piping and druming mixes；

(3) 96 porocyte culture plates are put into 37 DEG C of 5%CO₂After reacting 1h in incubator, by 50 hole μ L/ of serum-virus mixed liquor Addition is covered in 96 orifice plates of Vero cell, adds DMEM cell culture fluid of 200 holes μ L/ containing 2%FBS after adsorbing 1h, and 37 DEG C 5%CO₂Incubator culture 96h, observes and records result day by day.Neutralization titer is calculated according to Reed-Muench method.

2 results and analysis

2.1 PCV2 indirect ELISA test results

PCV2 antibody level variation line chart is shown in Figure 10.As shown in Figure 10: compared with DMEM control group, recombinant virus rPRV- gE^-/TK^-/ORF2⁺Group and PCV2 subunit vaccine immune group antibody level exempt to increase rapidly in 3 weeks one, antibody after exempting from two Level, which is presented, continues ascendant trend；Recombinant virus rPRV-gE^-/TK^-/ORF2⁺Immune group and PCV2 subunit vaccine immune group are anti- The horizontal variation tendency of body is consistent, and two groups of antibody levels are not much different after exempting from two.

2.2 PRV neutralize antibody titers are horizontal

Each anti-PRV neutralizing antibody level of immune group mouse is shown in Figure 11.Figure 11 is shown: DMEM immune group and PCV2 subunit vaccine are exempted from Epidemic disease group does not detect anti-PRV neutralizing antibody, rPRV-gE^-/TK^-/ORF2⁺Immune group is exempted from PRV live vaccine immune group in head Anti- PRV neutralizing antibody is produced within the 2nd week afterwards, and neutralizing antibody level difference is not significant (P > 0.05)；After two exempt from, rPRV- gE^-/TK^-/ORF2⁺Immune group and PRV live vaccine immune group neutralizing antibody titers highest can reach 1: 64.

Sequence table

<110>Agricultural University Of He'nan

<120>a kind of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain and its construction method

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<213>artificial sequence (Artificial Sequence)

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<213>artificial sequence (Artificial Sequence)

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<213>artificial sequence (Artificial Sequence)

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tttcttcccc caggaggggg ctcaaacccc cgctctgtgc cctttgaata ctacagaata 300

agaaaggtta aggttgaatt ctggccctgc tccccgatca cccagggtga caggggagtg 360

ggctccagtg ctgttattct agatgataac tttgtaacaa aggccacagc cctcacctat 420

gacccctatg taaactactc ctcccgccat accataaccc agcccttctc ctaccactcc 480

cgctacttta cccccaaacc tgtcctagat tccactattg attacttcca accaaacaac 540

aaaagaaatc agctgtggct gagactacaa actgctggga atgtagacca cgtaggcctc 600

ggcactgcgt tcgaaaacag tatatacgac caggaataca atatccgtgt aaccatgtat 660

gtacaattca gagaatttaa tcttaaagac cccccactta accctaagct t 711

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ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggctagctag ttattaatag 420

taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 480

acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 540

acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 600

ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 660

attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 720

gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 780

ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 840

caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 900

tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 960

tatataagca gagctggttt agtgaaccgt cagatccgaa ttcgagctcg gtacccgggg 1020

atcctctaga gtcgacctgc aggcatgcaa gcttggcact gtgccttcta gttgccagcc 1080

atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 1140

cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 1200

ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 1260

tggggatgcg gtgggctcta tggctactag tgagcttggc gtaatcatgg tcatagctgt 1320

ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 1380

agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 1440

tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 1500

cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 1560

gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 1620

ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 1680

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 1740

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 1800

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 1860

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 1920

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 1980

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 2040

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 2100

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 2160

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 2220

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 2280

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 2340

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 2400

agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 2460

ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 2520

gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 2580

catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 2640

cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 2700

cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 2760

gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 2820

tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 2880

gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 2940

tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 3000

gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 3060

gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 3120

taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 3180

tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 3240

ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 3300

taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 3360

tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 3420

aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 3480

ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c 3531

<210> 9

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

ccggaattcc tcctcctcgc cgccctgacc ctgg 34

<210> 10

<211> 67

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

ggactagtat aacttcgtat aatgtatgct atacgaagtt atcggacgga gataaaacgc 60

cacccac 67

<210> 11

<211> 67

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

ggactagtat aacttcgtat agcatacatt atacgaagtt atcctccgtc gacatggaca 60

cgtttga 67

<210> 12

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

cccaagcttc gtccagggcg tcggcgtccg tcag 34

<210> 13

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

ccggaattcc ggcgagcacg ctgtggccct ccag 34

<210> 14

<211> 67

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ggactagtat aacttcgtat aatgtatgct atacgaagtt attccgctgc cacaaccgct 60

tctacga 67

<210> 15

<211> 67

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

ggactagtat aacttcgtat agcatacatt atacgaagtt atgatggaga ccgcgacgga 60

ggcaacg 67

<210> 16

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cccaagcttc agcgagacgg cgcacggcga gagg 34

<210> 17

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

ttggatccct ttccgccgag acgaccc 27

<210> 18

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

cggaagcttc ggcgggtggt agatgcag 28

<210> 19

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gcgtactggc gcactctg 18

<210> 20

<211> 16

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

acgccgtcgt cgttgc 16

Claims (3)

1. a kind of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain, it is characterised in that: the Recombinant Swine Pseudorabies virus strain is rPRV-gE^-/TK^-/ORF2⁺。

2. a kind of structure of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain as described in claim 1 Construction method, which is characterized in that construction step is as follows:

(1), the building of recombinant plasmid pTK-ORF2

(1.1), the primer of PCR amplification ORF2, CMV-ORF2-poly A, TKU-CMV-ORF2-polyA-TKL are designed, respectively Are as follows:

F1: as shown in SEQ ID No.1；R1: as shown in SEQ ID No.2；

F2: as shown in SEQ ID No.3；R2: as shown in SEQ ID No.4；

F3: as shown in SEQ ID No.5；R3: as shown in SEQ ID No.6；

Wherein: F1 and R1 identifies ORF2 genetic fragment for PCR, and F2 and R2 are used to expand CMV-ORF2-polyA genetic fragment, F3 and R3 is for expanding TKU-CMV-ORF2-polyA-TKL genetic fragment；

(1.2), the synthesis of 2 gene of PCV2 genome ORF and pZJ-1 carrier

It is respectively synthesized ORF2 gene and pZJ-1 carrier, ORF2 gene order is as shown in SEQ ID No.7, pZJ-1 carrier sequence As shown in SEQ ID No.8；

(1.3), construction recombination plasmid pZJ-ORF2

Distinguish double digestion ORF2 genetic fragment and pZJ-1 carrier using Kpn I, Hind III, ORF2 after recycling digestion and PZJ-1 passes through T₄DNA ligase is attached, and carries out plasmid conversion later, bacterium solution PCR screening positive clone will be with positive gram Grand opposite bacterium solution expands culture, and extracts plasmid, which is named as pZJ-ORF2；

(1.4), Nhe I and Spe I double digestion recombinant plasmid pZJ-ORF2

Using the DNA of recombinant plasmid pZJ-ORF2 as template, using F2 and R2 as primer, pcr amplification reaction is carried out, after the completion of amplification, Agarose gel electrophoresis simultaneously recycles pcr amplification product；Nhe I and Spe I double digestion pcr amplification product recycles digestion products, obtains Obtain CMV-ORF2-polyA genetic fragment；

(1.5), Spe I single endonuclease digestion pTK carrier and dephosphorylation process

Using Spe I single endonuclease digestion pTK carrier, dephosphorylation, purifies again after purification；

(1.6), CMV-ORF2-polyA genetic fragment and the processed pTK carrier digestion products of step (1.5) pass through T₄DNA connects It connects enzyme to be attached, carries out plasmid conversion later, bacterium solution PCR screening positive clone carries out the bacterium solution opposite with positive colony Expand culture, extract plasmid, which is named as pTK-ORF2；

(2), recombinant virus rPRV-gE^-/TK^--ORF2⁺Building

(2.1), using recombinant plasmid pTK-ORF2 as template, using F3 and R3 as primer, pcr amplification reaction, Ago-Gel are carried out Electrophoresis simultaneously recycles, and obtains TKU-CMV-ORF2-PloyA-TKL genetic fragment；

(2.2), PRV gene-deleted strain rPRV-gE is extracted^-/TK^-/EGFP⁺The genomic DNA of recombinant virus；

(2.3), by rPRV-gE^-/TK^-/EGFP⁺Genome and TKU-CMV-ORF2-PloyA-TKL genetic fragment cotransfection are extremely 293T cell, plaque purification obtain recombinant virus rPRV-gE^-/TK^-/ORF2⁺。

3. the building side of expression porcine circovirus 2 type ORF2 genetic recombination porcine pseudorabies virus strain as claimed in claim 2 Method, it is characterised in that: in step (1.5), the pTK carrier is the plasmid pTK that applicant voluntarily constructs；In step (2.2), institute State PRV gene-deleted strain rPRV-gE^-/TK^-/EGFP⁺The recombination fluorescence virus HN-QYY-gE voluntarily constructed for applicant^-/TK^-/EGFP⁺；Plasmid pTK and recombination fluorescence virus HN-QYY-gE^-/TK^-/EGFP⁺Construction step it is as follows:

S1, missing gE gene:

S1.1, the variant HN-QYY that deposit number is CGMCC No.15192 is seeded to the vero cell for covering with single layer, when When 80% or more lesion occurs in cell, virus liquid, multigelation are harvested, centrifugation takes supernatant, and phenol chloroform method extracts variation Strain HN-QYY virus genom DNA, saves backup；

S1.2, using variant HN-QYY virus genom DNA as template, utilize primer gEL-f/gEL-r and gER-f/gER-r point Other PCR amplification gE gene upstream and downstream homology arm gEL and gER, purified pcr product after agarose gel electrophoresis identification, measurement DNA contain Amount, saves backup；

Wherein, primer sequence is respectively as follows:

GEL-f: as shown in SEQ ID No.9；GEL-r: as shown in SEQ ID No.10；

GER-f: as shown in SEQ ID No.11；GER-r: as shown in SEQ ID No.12；

S1.3, EcoR I and the bis- enzymes of Spe I double digestion gEL, Spe I and Hind III double digestion gER, EcoR I and Hind III PMD18-T carrier is cut, gEL, gER, pMD18-T after recycling digestion；

S1.4, gEL after the recovery and gER are connected on pMD18-T after the recovery, carry out plasmid conversion, bacterium solution PCR sieve later Positive colony is selected, the bacterium solution opposite with positive colony is expanded culture, extracts plasmid, which is named as pgE；

S1.5, Spe I single endonuclease digestion plasmid pgE, dephosphorylation, purifies again after purification；

S1.6, Nhe I and Spe I double digestion pEGFP-N1 carrier, the pEGFP-N1 after recycling digestion；By pEGFP- after the recovery N1 is connected on the pgE after dephosphorylation, carries out plasmid conversion later, bacterium solution PCR screening positive clone will be with positive colony phase Pair bacterium solution expand culture, extract plasmid, which is named as pgE-EGFP；

S1.7, building recombination fluorescence virus HN-QYY-gE^-/EGFP⁺

By variant HN-QYY virus genom DNA, pgE-EGFP cotransfection obtained by step S1.1, to 293T cell, plaque is pure Change, until green fluorescence occur in all cytopathies, obtains recombination fluorescence virus HN-QYY-gE^-/EGFP⁺；

S1.8, removal green fluorescence gene

By HN-QYY-gE^-/EGFP⁺It is Plaque-purified with pcGlobin2-Cre plasmid co-transfection to 293T cell, until all thin Born of the same parents' lesion without fluorescence, obtains porcine pseudorabies virus variant HN-QYY-gE^-；

S2, missing TK gene:

S2.1, by variant HN-QYY-gE obtained by step S1.8^-It is seeded to the vero cell for covering with single layer, when cell occurs 80% When the above lesion, virus liquid, multigelation are harvested, centrifugation takes supernatant, and phenol chloroform method extracts variant HN-QYY-gE^- Virus genom DNA saves backup；

S2.2, with variant HN-QYY-gE^-Virus genom DNA is template, utilizes primer TKL-f/TKL-r and TKR-f/TKR- R distinguishes PCR amplification TK gene upstream and downstream homology arm TKL and TKR, purified pcr product after agarose gel electrophoresis identification, measurement DNA content saves backup；

Wherein, primer sequence is respectively as follows:

TKL-f: as shown in SEQ ID No.13；TKL-r: as shown in SEQ ID No.14；

TKR-f: as shown in SEQ ID No.15；TKR-r: as shown in SEQ ID No.16；

S2.3, EcoR I and the bis- enzymes of Spe I double digestion TKL, Spe I and Hind III double digestion TKR, EcoR I and Hind III PMD18-T carrier is cut, TKL, TKR, pMD18-T after recycling digestion；

S2.4, TKL after the recovery and TKR are connected on pMD18-T after the recovery, carry out plasmid conversion, bacterium solution PCR sieve later Positive colony is selected, the bacterium solution opposite with positive colony is expanded culture, extracts plasmid, which is named as pTK；

S2.5, Spe I single endonuclease digestion plasmid pTK, dephosphorylation, purifies again after purification；

S2.6, Nhe I and Spe I double digestion pEGFP-N1 carrier, the pEGFP-N1 after recycling digestion；By pEGFP- after the recovery N1 is connected on the pTK after dephosphorylation, carries out plasmid conversion later, bacterium solution PCR screening positive clone will be with positive colony phase Pair bacterium solution expand culture, extract plasmid, which is named as pTK-EGFP；

S2.7, building recombination fluorescence virus HN-QYY-gE^-/TK^-/EGFP⁺

By variant HN-QYY-gE obtained by step S2.1^-Virus genom DNA, pTK-EGFP cotransfection to 293T cell, plaque Purifying obtains recombination fluorescence virus HN-QYY-gE until green fluorescence occur in all cytopathies^-/TK^-/EGFP⁺。