CN109207438A

CN109207438A - Porcine pseudorabies virus velogen strain and its preparing the application in inactivated vaccine

Info

Publication number: CN109207438A
Application number: CN201810952429.9A
Authority: CN
Inventors: 崔宇润
Original assignee: Beijing Kanggu Biotechnology Co ltd
Current assignee: Beijing Kanggu Biotechnology Co ltd
Priority date: 2018-08-20
Filing date: 2018-08-20
Publication date: 2019-01-15

Abstract

The invention discloses a kind of porcine pseudorabies virus velogen strain and its application in inactivated vaccine is being prepared, is belonging to pharmaceutical technology field.Porcine pseudorabies virus velogen strain of the present invention, it is named as BJ, the strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, culture presevation number are as follows: CGMCC No.15911.It is demonstrated experimentally that primiparity health pig is immunized for preventing porcine pseudorabies in the swine pseudorabies vaccine being prepared using the strain, have many advantages, such as that safety, generation antibody is fast, duration of immunity is lasting.And it can be used for constructing porcine pseudorabies virus and attack poison morbidity model and the dual-gene gene-deleted strain vaccine strain of porcine pseudorabies virus gE/gI, can match with corresponding DIVA (infection and immune animal can be distinguished) strategy.A kind of porcine pseudorabies virus velogen strain of the invention will be with a wide range of applications in porcine pseudorabies prevention and control field.

Description

Porcine pseudorabies virus velogen strain and its preparing the application in inactivated vaccine

Technical field

The present invention relates to a kind of porcine pseudorabies virus velogen strain and its preparing the application in inactivated vaccine.The invention belongs to In pharmaceutical technology field.

Background technique

Porcine pseudorabies are a kind of urgency of high mortality caused by Pseudorabies virus (Pseudorabiesvirus, PRV) Sexually transmitted disease.Pseudorabies virus belongs to herpetoviridae (Herpesviridae), herpesvirus suis category.Pseudorabies virus can be felt A variety of hosts are contaminated, but pig is main natural host, storage person and the disseminator of the virus.Porcine pseudorabies virus can infect difference The pig of age bracket, but the most serious with pregnant sow and suckling pig infection: lead to pregnant sow miscarriage, stillborn foetus and mummy Tire；There is nervous symptoms, paralysis, failure death in suckling pig, and the death rate is almost up to 100%.

PRV is α herpetoviridae, Varicellavirus member, and genome is linear distrand DNA, by unique long section (area UL), unique short section (area US), internal repeat (IR) and end are reversed repetitive sequence (TR) and are constituted.PRV gene Containing about 70~100 coding protein genes of group, wherein the genes such as gE, gI, gC, TK, PK are related to PRV virulence and increase for PRV Grow dispensable gene.It gE, gI and PRV Neural invasion and propagates in nervous system related, is sent out by the heterodimer of formation The effect of waving.

From pseudo- mad dog gene-deleted vaccine is begun to use at the end of the 20th century, pseudorabies disease incidence is decreased obviously on China pig farm. But since 2011, many large-scale pig farms being immunized using gE gene deleted live vaccine occur sow produce weak son, stillborn foetus, There are the clinical symptoms of the doubtful mad dog of puppet such as nervous symptoms and death and generate serious economic loss in miscarriage, piglet.It is related to grind Study carefully separation identification, gE gene sequencing and blood that the clinical sample that pig farm is immunized to multiple provinces in scholar has carried out the wild poison of PRV As a result clear neutralization test tentatively shows that newly separating the more previous strain of PRV prevalence strain occurs obvious variation, the cause to mouse, pig Characteristic of disease is remarkably reinforced；There is pruritis, thermophilic viscera tissue enhancing in morbidity piglet.Therefore, it develops and sends out prevalence again for this The vaccine of strain, it is very necessary.

Summary of the invention

Epidemic disease is inactivated the purpose of the present invention is to provide a kind of newfashioned porcine pseudorabies virus velogen strain and its in preparation Application in seedling.

In order to achieve the above object, present invention employs following technological means:

Inventor is sent out with ST cell from the doubtful porcine pseudorabies of the pig farm inspection on the ground such as Hebei, northeast, Beijing Separation obtains 6 plants of Pseudorabies virus in sick pig pathological material of disease, carries out PCR identification and gB, gE gene sequencing point to 6 plants of PRV viruses Analysis, as a result, it has been found that 6 strain virus are closer with the variation strain virus affiliation separated in recent years, it is variant porcine pseudorabies Poison.Animal Orthogonal Rotational Regressive Tests show that 6 strain virus can cause mouse, family's rabbit invasion or death.Through piglet inoculation test, separation BJ plants of virulence are most strong.Show that its immunogenicity is good by the test result in later period, can be used as the candidate strain of vaccine research.

A kind of porcine pseudorabies virus velogen strain proposed by the present invention, is named as BJ, and classification naming is porcine pseudorabies disease Poison, the strain are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is in Chaoyang District, Beijing City No. 3 Institute of Microorganism, Academia Sinica of institute of North Star West Road 1, culture presevation number are as follows: CGMCC No.15911, preservation day Phase is on July 11st, 2018.

Further, the invention also provides the porcine pseudorabies virus velogen strains to prepare porcine pseudorabies inactivation Application in vaccine.And

The porcine pseudorabies virus velogen strain establish porcine pseudorabies virus attack poison morbidity model in application.

Wherein, it is preferred that the porcine pseudorabies virus attacks poison morbidity model in vaccine potency inspection.

Wherein, it is preferred that carry out attacking poison using the pure culture of the porcine pseudorabies virus velogen strain.

In order to match with corresponding DIVA (can distinguish infection and immune animal) strategy, further, the present invention is also The porcine pseudorabies virus velogen strain is proposed in preparing the dual-gene deletion of vaccine strain of porcine pseudorabies virus gE/gI Using.

Wherein, it is preferred that the dual-gene gene-deleted strain vaccine strain of the porcine pseudorabies virus gE/gI is pseudo- in the pig On the basis of hydrophobin velogen strain, obtained by the dual-gene building of knockout gE and gI.

Further, the invention also provides a kind of swine pseudorabies vaccines, the present invention after containing inactivation The porcine pseudorabies virus velogen strain.

Wherein, it is preferred that take sterilizing 206 VG adjuvant of ISA, 1 parts by volume, the porcine pseudorabies virus after inactivation is added is strong 1 parts by volume of strain virus liquid, mixing, stirs at low speed 30~60 minutes, vaccine is made.

Wherein, it is preferred that before inactivation, every milliliter of viral level answers >=10^7.5TCID50。

Wherein, it is preferred that the inactivation is will to examine qualified virus liquid, is slowly added to final concentration of 0.05v/v% Binary ethylenimine (BEI) solution, it is stirring while adding, be uniformly mixed, set 30 DEG C inactivate 48 hours, be added final concentration 2v/v% sulphur Sodium thiosulfate solution terminates inactivation.

It is demonstrated experimentally that primiparity health pig is immunized for preventing in the swine pseudorabies vaccine being prepared using the present invention Porcine pseudorabies have many advantages, such as that safety, generation antibody is fast, duration of immunity is lasting.

Detailed description of the invention

Fig. 1 is the PCR qualification result of PRV-Batha-K61 and PRV-BJ；

M:DL2000 Marker；1: negative control；2: positive control；3:PRV-Batha-K61；4:PRV-BJ；

Fig. 2 is pEGFP-C1 Reconstruc-tion policy；

Fig. 3 is that the identification of plasmid double digestion is transformed in pMEGFP-C1；

M:DNAMarker；1,2:pEGFP-C1 plasmid；3:pMEGFP-C1

Fig. 4 is effect homology arm PCR amplification；

M:DNA Marker；R: right homology arm；L: left homology arm；

Fig. 5 is the identification of recombinant plasmid pMD- Δ gE/gI-L-EGFP-R double digestion；

1:pMD- Δ gE/gI-L-EGFP-R；M:DNA Marker

Fig. 6 is pMD- Δ gE/gI-L-EGFP-R transfer vector activity identification；

A: transfection pEGFP-C1 plasmid 293T cell；B: blank control 293T cell；C: transfection recombinant plasmid pMD- Δ gE/ The 293T cell of gI-L-EGFP-R

Fig. 7 is the identification of recombinant plasmid pMD- Δ gE/gI-L-R double digestion；

1:pMD- Δ gE/gI-L-R；M:DNA Marker

Fig. 8 is PRVBJ- Δ gE/gI-EGFP qualification result；

A: negative control；B:PRVBJ- Δ gE/gI-EGFP virus infected cell；

Fig. 9 is the identification of recombinant plasmid pMD- Δ gE/gI-L-R double digestion；

1:pMD- Δ gE/gI-L-R；M:DNA Marker

Figure 10 is gE/gI plants of Fluorescence Identification results of PRVBJ- Δ；

Figure 11 is PRVBJ- Δ gE/gI PCR qualification result；

M:DNAMarker；1:PRV-gEf/PRV-gEr；2:PRV-gBf/PRV-gB；3:PRV ID E/I up/PRV ID E/I down

The piglet that Figure 12 is 14 days after inoculation.

A: control group；B: experimental group

Specific embodiment

The advantages of the invention will now be further described with reference to specific embodiments, the invention and feature will be with retouching It states and apparent.But examples are merely exemplary for these, does not constitute any restrictions to the range of the invention.Ability Field technique personnel should be understood that can be to technical solution of the present invention under the spirit and scope without departing from the invention Details and form are modified or are replaced, but the replacement of these modifications is each fallen in the protection scope of the invention.

The separation of BJ plants of 1 porcine pseudorabies virus of embodiment is identified

1 materials and methods

1.1 pathological material of disease

Pathological material of disease used is tested from the different regions such as Hebei, northeast, Beijing, the piglet on 6 morbidity pig farms or the mummification of fetus Youngster, morbidity piglet show following clinical symptoms: body temperature increases, spirit is tired, severe is thin, movement is uncoordinated, shakes, spasm Deng.Dissect takes the brain tissue of every pig, the heart, liver, spleen, lung, kidney, tonsillotome as pathological material of disease；Brain tissue is as a sample, nothing Brain tissue person takes each internal organs mixing as pathological material of disease；It is thorough with mortar grinder after pathological material of disease is shredded, it is added after a certain amount of PBS instead Multiple freeze thawing 3 times, 4000r/min are centrifuged 15min, take supernatant, through 0.22 μm of -20 DEG C of postposition of membrane filtration degerming or less preservations or Inoculating cell carries out a point poison immediately, and pathological material of disease is taken to make PCR measurement.

1.2 cells and main agents

PK15 cell, ST cell and VERO cell, are saved by this laboratory, the separation for Pseudorabies virus；DMEM Purchased from GIBCO company；DL2000 DNA Marker is purchased from precious bioengineering (Dalian) Co., Ltd；PCR kit is purchased from Shandong Great Zheng Medical Treatment Equipment Co., Ltd；DNA extraction kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd.；Glue recycling examination Agent box is Axygen Products；Cell culture fluid is inactivated fetal bovine serum, each 100U (μ g)/mL of mycillin containing 8% DMEM；Agarose is Spanish Products；The reagent used in other experiments is import or domestic analytical grade reagent.

1.3 experimental animal

4~6 week old mouse are purchased from Zhejiang University of Traditional Chinese Medicine；1.5~3.0Kg rabbit is purchased from Zhejiang University of Traditional Chinese Medicine；2~ 3 week old piglets are purchased from periphery raiser.

1.4 virus purification

The PK15 cell, ST cell and VERO cell of good single layer will be grown up to, has discarded culture solution, then will grind pathological material of disease And the good filtrate of filtration sterilization is inoculated into cell monolayer, 37 DEG C adsorb 1 hour, discard, cell maintenance medium is then added, set 37 DEG C, contain 5%CO₂Incubator culture was observed to 96 hours.If any CPE, then -20 DEG C of refrigerators, 37 DEG C of water-bath freeze thawing 3 times harvests are set. Such as without CPE, then continue 3 generation of blind passage.

1.5PCR measurement

According to pertinent literature, designs the highly conserved region of gB, gE, gG gene and design three pairs of primers, it is contemplated that amplified fragments are big Small is respectively 431,316 and 202bp.Its sequence is as shown in table 1, extracts DNA according to a conventional method, carries out PCR amplification, amplified production Gene sequencing company is sent to be sequenced.

The primer sequence of the amplification each gene of PRV of table 1

F: forward primer；R: reverse primer.

1.6 viral purification

Virus is serially diluted again with DMEM maintaining liquid 10 to 10^-10, take 10^-3~10^-10The suspension inoculation of 8 dilutions is Grow up to the 96 hole Microtitration plates of ST cell of single layer, each dilution is inoculated with 8 holes, separately sets 8 holes and does not connect poison conduct pair According to.Set 37 DEG C, containing 5%CO₂Incubator culture 5 days, control wells cellular morphology should be good.Highest dilution lesion hole is taken, is collected Supernatant, as first time purified virus.

The ST cell for taking the inoculation of first time purified virus to grow up to single layer is proliferated, and when lesion reaches 80% or so, is frozen Melt 3 times, supernatant is taken to be serially diluted again with DMEM maintaining liquid 10, inoculation has grown up to the 96 hole Micro-CPE neutralization test of ST cell of single layer Plate, each dilution are inoculated with 8 holes, separately set 8 holes and do not connect poison as control.Set 37 DEG C, containing 5%CO₂Incubator culture 5 days, Control wells cellular morphology should be good.Highest dilution lesion hole is taken, supernatant, as second of purified virus are collected.

According to the method described above by viral repurity 1 time, the virus that obtained purified virus as purifies three times.Measurement TCID₅₀Afterwards, -70 DEG C are set to save backup.

The measurement of 1.7 viral levels

It uses the maintaining liquid containing 2% newborn bovine serum to make 10 times in venom to be serially diluted, take 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-88 dilutions, inoculation grows up to good 96 hole microtest plate of single layer ST cell respectively, and each dilution connects 8 holes of kind, every 100 μ l of hole.Add the 100 μ l of cell culture fluid containing 2% newborn bovine serum in every hole.It sets simultaneously and does not connect 8 holes of poison control, Set 37 DEG C, containing 5%CO₂It cultivates, observe 96~120 hours in incubator, record the hole count of cytopathy (CPE).By Reed- Muench method calculates TCID₅₀。

1.8 animal Orthogonal Rotational Regressive Tests

1.8.1 rabbit inoculation test

Rabbit 3 of 1.5~3.0Kg, every difference intramuscular inoculation virus purification culture 0.5ml are taken, if compareing 2, Rabbit spirit, diet and clinical manifestation are observed after inoculation.

1.8.2 mouse inoculation is tested

Take 10 4~6 week old or so mouse, 5 every intraperitoneal inoculation virus purification culture 0.2ml, another 5 back ridges Nearby 0.2ml is subcutaneously injected in column, and mouse spirit, diet and clinical manifestation are observed after inoculation.

1.8.3 piglet inoculation test

Take 2~3 week old piglet 3, every collunarium and each 3ml of intramuscular injection.It sets simultaneously and does not attack poison control pig 2.Inoculation Observation piglet spirit, diet and clinical manifestation afterwards.To the dead timely dissect of pig, the 7th day dissect after morbid pig inoculation is acquired flat Peach body isolated viral.

2 results

2.1 virus purification

Treated filtrate is inoculated with ST cell, HB plants, BJ plants, HB2 plants, TJ1 plants, TJ2 plants, JL plants it is after inoculation or blind It passed for 1~3 generation, typical cells lesion occurs, show as the contracting of cell circle, fall off, and control cell is still able to maintain intact monolayer.

2.2 viral purification

For 6 strain virus of separation respectively through 3 generation of limiting dilution assay clone purification, viral level reaches 10^6.0~8.5TCID₅₀/ml。 Concrete outcome is shown in Table 2.

2 purified virus poison valence measurement result of table

2.3 PCR measurement results

PCR identification is carried out to PRV-BJ and PRV-Batha-K61 vaccine strain, is as a result amplified for PRV-Batha-K61 plants Two specific bands of 431bp (gB) and 202bp (gG), and PRV-BJ plants amplified 431bp (gB), 316bp (gE) and The specific electrophoretic band (Fig. 1) of 3 of 202bp (gG).It demonstrates PRV-BJ and contains gE gene, not with Batha-K61 vaccine strain With (gE missing), for street strain currently popular.HB plants, HB2 plants, TJ1 plants, TJ2 plants, JL plants, which are detected, through PCR is similarly wild poison Strain.

2.4 animal Orthogonal Rotational Regressive Tests

2.4.1 rabbit inoculation test

After 6 plants of separation poison inoculation rabbit, cause rabbit dead, dead rabbit occur surprise itch, the symptoms such as the inoculation position that bites, Illustrate 6 plants separation be it is virulent, the results are shown in Table 3.

3 rabbit inoculation test result of table

2.4.2 mouse inoculation is tested

After the malicious Mice Inoculateds of 6 plants of separation, cause dead mouse, illustrate 6 plants of separation poison be it is virulent, the results are shown in Table 4.

4 mouse inoculation test result of table

2.4.3 piglet inoculation test

After 6 plants of separation poison inoculation piglets, piglet morbidity and dead degree are different, illustrate that 6 plants of separation are virulent.Wherein BJ Strain dead 3/3, virulence is most strong, the results are shown in Table 5.

5 piglet inoculation test result of table

3 conclusions

Several plants of PRV of separation, observed by pathological anatomy, virus purification, cell culture, malicious valence measurement, morphology of virus, The methods of PCR identification, sequence analysis, animal Orthogonal Rotational Regressive Tests, it was demonstrated that be virulent.Wherein, BJ plants of malicious valences are higher, and animal returns examination It tests and is shown to be one plant of velogen strain, show that its immunogenicity is good by the test result in later period, candidate's poison as vaccine research Strain.The strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is in Chaoyang District, Beijing City No. 3 Institute of Microorganism, Academia Sinica of institute of North Star West Road 1, culture presevation number are as follows: CGMCC No.15911, preservation day Phase is on July 11st, 2018.

The preparation of embodiment BJ plants of inactivated vaccines of 2 porcine pseudorabies virus velogen strain and efficacy test

1 seed culture of viruses

It is BJ plants of porcine pseudorabies virus velogen strain that vaccine seed culture of viruses and inspection, which are manufactured, with seed culture of viruses.Without bacterium, mould, Mycoplasma and exogenous virus pollution.

The manufacture of 2 vaccines and the inspection of semifinished product

2.1 productions are prepared with seed culture of viruses

2.1.1 seed culture of viruses is bred

The ST cell for single layer of having grown up is discarded into growth-promoting media, seed culture of viruses BJ plants is inoculated with by 2% amount, is added and contains 2% new born bovine The DMEM cell culture fluid of serum, set 37 DEG C cultivate 2~3 days, the harvest when lesion occurs in 75% or more cell, set -15 DEG C with Lower freeze thawing 1 time, quantitative separating indicate harvest date, seed culture of viruses algebra (should be no more than for 5 generations) etc., -35 DEG C or less preservations, storage life It is 6 months.

The preparation of 2.2 cells

ST cell is taken out from liquid nitrogen container, 37 DEG C of water-bath fast melts, 1000r/mim is centrifuged 5 minutes, with containing 10% new life The cell culture fluid suspension cell of cow's serum is set 37 DEG C, is cultivated in incubator containing 5%CO2, when growing up to good single layer, uses 0.25% trypsin solution vitellophag passes on Multiplying culture.

The breeding of 2.3 seedling venom

2.3.1 inoculation

The cell spinner bottle for having grown up to single layer is taken, growth-promoting media is discarded, production seed culture of viruses is inoculated with by 2% amount, is added new containing 2% The DMEM cell culture fluid of raw cow's serum, sets 37 DEG C of rotating and culturings, and revolving speed is 8~10 turns/hour.

2.3.2 harvest

CPE is observed after inoculation daily, the harvest when CPE reaches 80% or more freeze thawing 1 time, is centrifuged or is removed with 1 μm of filter core Cell fragment harvests antigen liquid.It sets -15 DEG C or less and saves and be no more than 1 month.

2.3.3 viral level measures

To the antigen liquid sampling after harvest, it is measured, every milliliter of viral level answers >=10^7.5TCID50。

2.4 inactivation

Qualified virus liquid will be examined, is slowly added to final concentration of 0.05% binary ethylenimine (BEI) solution, side edged Stirring is uniformly mixed, and is set 30 DEG C and is inactivated 48 hours, and 2% hypo solution of final concentration is added and terminates inactivation.Antigen after inactivation 2~8 DEG C of preservations are placed, should be no more than 1 month.

2.5 the inspection of semifinished product

2.5.1 inactivation is examined

Virus liquid after taking inactivation has grown up to the ST cell of single layer after absorption 1 hour in 5% ratio inoculation and has changed into and contain The cell maintenance medium of 2% newborn bovine serum is set 37 DEG C of cultures and is observed 5.Harvest cell culture, freeze thawing, on ST cell again It blind passage 1 time, should be without CPE.

2.5.2 steriling test

It is carried out by existing " Chinese veterinary pharmacopoeia " annex, regulation should be met.

2.6 vaccine preparation

1 part of 206 VG adjuvant of ISA of sterilizing is taken, adds and examines qualified 1 part of inactivation antigen, mixing stirs at low speed 30~60 points Vaccine is made in clock.

2.7 packing

By the sterile quantitative separating of vaccine in sterilizing vaccine bottle, seal, adhesive label.Set 2~8 DEG C of preservations.

4 product inspections

Dosage form: it is in W/O/W dosage form (w/o/w).A cleaning suction pipe is taken, draws a small amount of vaccine drop in cleaning cold water meter Face should be spread in cloud.

Stability: drawing vaccine 10ml and be added in centrifuge tube, with 3000r/min centrifugation 15 minutes, should not be demulsified, and water 0.5ml should be no more than by being mutually precipitated.

Viscosity: it tests by existing " Chinese veterinary pharmacopoeia " annex, regulation should be met.

5 efficacy tests

5.1 piglet immunologicals attack poison

With the susceptible piglet of 2~3 week old health, (PCR antigen is detected as feminine gender；Porcine pseudorabies serum neutralize antibody titers are not Higher than 1: 4) 10, being randomly divided into 2 groups, every group 5.One group is immune group, and each musculi colli of every piglet vaccinates 2ml (10^7.5TCID50/ml), same dosage booster immunization 1 time on the 21st after being immunized, another group is not to be inoculated with control group, in the same terms Lower isolated rearing.After two exempt from 21, all pigs BJ plants of virulent intramuscular injection of porcine pseudorabies virus and each 3.0ml of collunarium (10^7.0TCID50/ml), every range estimation is warm and observes 14.Attacking poison control pig should all fall ill；Immune swine at least protects 4.

5.2 mouse immunes attack poison

It is each that vaccine 0.3ml (10 is subcutaneously injected with 17~22g of weight mouse 10^7.5TCID50/ml), reinforce exempting from after 2 weeks Epidemic disease is primary.After observing 2 weeks, together with the identical control mice of condition 5, porcine pseudorabies virus BJ plants of 0.2ml is subcutaneously injected and (contains 50LD50), it observes 10.Control group should be all dead, and immune group is at least survived 8.

BJ plants of 3 porcine pseudorabies virus velogen strain of embodiment are preparing the dual-gene gene-deleted strain of porcine pseudorabies virus gE/gI Application in vaccine strain (BJC plants)

1 materials and methods

1.1 viruses and cell

PRV BJ strain virus is isolated by embodiment 1；ST cell and Vero cell are saved by this laboratory.DH5 α impression State cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd.

1.2 plasmid

PEGFP-C1 is saved by this laboratory.PMD18-T carrier is purchased from Takara.

1.3 design of primers

Primer is as shown in table 6 below.

The primer applied in 6 PRV gE/gI of table knockout

1.4 construction strategy

PCR amplification is carried out using the primer of amplification PRV or so homology arm to obtain the left and right homology arm of building transfer vector. PEGFP-C1 is subjected to double digestion using Bsp EI and BamH I to remove unused restriction enzyme site simultaneously, utilizes Ase I and Mlu I digestion goes out EGFP expressed intact box.By being sequentially inserted into pMD18-T carrier for left homology arm-EGFP expression cassette-right homology arm, To obtain recombinant transfer vector pMD- Δ gE/gI-L-EGFP-R.

The transformation of 1.5 pEGFP-C1 plasmids

In order to delete cloning site extra in pEGFP-C1 plasmid, Bsp EI and BamH I restriction enzyme enzyme is utilized PEGFP-C1 plasmid is cut, large fragment is recycled.The large fragment is converted into DH5 α competent cell, using Escherichia coli enzyme system by matter Grain completion.Plasmid is extracted, is verified with EcoRI and Ase I, successful plasmid is transformed and is named as pMEGFP-C1 (Fig. 2).

The amplification of 1.6 or so homology arms

Utilize PRV Del E/I-L up/PRV Del E/I-L down and PRV Del E/I-R up/PRV Del E/ I-R down two carries out PCR amplification, reaction system such as table 7 by template of BJ plants of DNA of PRV respectively to primer.Reaction condition is 95℃10min；95 DEG C of 60s, 65 DEG C of 45s, 72 DEG C of 90s, 35 circulations；72℃10min.Gel electricity is carried out on 1% Ago-Gel Swimming detection, recycles segment of corresponding size.

7 or so homology arm PCR amplification system of table

The building of 1.7 pMD- Δ gE/gI-L-EGFP-R transfer vectors

The pMEGFP-C1 plasmid of transformation is subjected to digestion with two restriction enzymes of Ase I and Mlu I, is recycled 1584bp size segment.Left homology arm is subjected to double digestion with EcoR I and Ase I；By right homology arm with Mlu I and Xho I into Row double digestion, the left and right homology arm after digestion is separately recovered.After pMD18-T carrier is carried out double digestion with EcoR I and Xho I Recycle large fragment.4 segments of recycling are attached with T4DNA ligase, convert Escherichia coli, choose bacterium, plasmid is extracted and uses EcoR I and Xho I carry out double digestion identification, should obtain the two of about 2.6kb and about 3.6kb after constructing successful plasmid double digestion A segment.Such as 8, the 37 DEG C of digestions of table of double digestion system are stayed overnight.

8 double digestion system of table

1.8 pMD- Δ gE/gI-L-EGFP-R transfer vector activity identifications

PMD- Δ gE/gI-L-EGFP-R transfer vector is linearized with EcoR I restriction enzyme.It will linearisation Plasmid according to Promega calcium phosphate transfection kit specification transfect Vero, cultivate 24 to 48 hours, see under the microscope It examines.

1.9 knock out the building of the transfer vector pMD- Δ gE/gI-L-R of EGFP expression cassette

Using PRV Del EGFP-L up/PRV Del EGFP-L down and PRV ID EGFP-R up/PRV in table 9 ID EGFP-R down two carries out PCR amplification to primer, by template of PRV DNA.PCR reaction condition and reaction system such as 1.6 Described in.Agarose gel electrophoresis detection is carried out using 1% Ago-Gel, recycles purpose band.L, R segment of recycling, point Double digestion is not carried out with EcoR I, Mlu I and Mlu I, Xho I.PMD18-T is subjected to double digestion, enzyme with EcoR I and Xho I Large fragment is recycled after cutting.3 DNA fragmentations of recycling are attached with T4DNA ligase, Escherichia coli is converted, chooses bacterium, are extracted Plasmid carries out double digestion identification with EcoRI and Xho I, and the plasmid of successful connection answers digestion to go out two of about 2.6kb and about 2kb DNA fragmentation.

The primer of the building pMD- Δ gE/gI-L-R transfer vector of table 9

The extraction of 1.10 PRV DNA

BJ plants of PRV are inoculated with the ST cell for growing up to single layer, when cytopathy reaches 70%~80%, abandon part and cultivate Base.Cell is scraped with cell scraper, centrifuge tube 2000rmp centrifugation 10min is transferred to, abandons supernatant.Cell precipitation is resuspended with TNE, so Extract total DNA with phenol/chloroform extraction method afterwards, dissolved with TE, be stored in -80 DEG C it is spare.

The rescue of 1.11 PRV BJ- Δ gE/gI-EGFP

The transfer vector pMD- Δ gE/gI-L-EGFP-R of building is linearized with EcoR I restriction enzyme.Line Property plasmid and BJ plants of PRV of DNA cotransfection degrees of fusion about 70% ST cell, cotransfection operation is referring to Promega phosphoric acid Calcium transfection reagent box operating instruction carries out.ST cell after transfection, sets in cell incubator and cultivates.After cytopathy to appear, Harvest virus, obtained viral nomenclature are BJ- Δ gE/gI-EGFP.

1.12 PRV BJ- Δ gE/gI-EGFP purifying

The BJ- Δ gE/gI-EGFP virus liquid of harvest is subjected to 10 times of gradient dilutions, dilutes 6 gradients.After dilution Poison disease vaccination is grown on the single layer ST cell of 6 porocyte culture plates, after adsorbing 1h, spreads plus contain 1% low melting point agar in each hole The MEM of sugar and 2%FBs, sets room temperature, to be inverted culture in agarose solidification postposition cell incubator.Poison is connect after 48 hours, aobvious Micro- microscopic observation, the plaque of label expression green fluorescence.The plaque of picking label is placed in serum-free DMEM, and -80 DEG C freeze repeatedly Melt 3 times.The plaque liquid inoculation ST cell of picking is subjected to next round plaque purification if still there is green fluorescence.

The PCR of 1.13 PRV BJ- Δ gE/gI-EGFP is identified

Referring to viral DNA rapidly extracting kit specification, PRV BJ- Δ gE/gI-EGFP DNA is extracted, with PRV ID E/I up/PRV ID E/I down, PRV-gBf/PRV-gBr, PRV-gEf/PRV-gEr, Jegfp F/Jegfp R4 respectively into Row PCR identification.PCR reaction system is 25 μ L:2 × GC BufferII, 12.5 μ L；dNTP mix(2.5mM/μL)4μL；On Swim primer (10 μM/μ L) 1 μ L；Downstream primer (10 μM/μ L) 1 μ L；0.25 μ L of LAtaq enzyme；PRV BJ-ΔgE/gI-EGFP DNA2μL；ddH₂O4.25μL。PRV ID E/I up/PRV ID E/I down、PRV-gBf/PRV-gBr、PRV-gEf/PRV- GEr primer PCR reaction condition are as follows: 95 DEG C of 10min；95 DEG C of 50s, 60 DEG C of 45s, 72 DEG C of 90s, 35 circulations；72℃10min.Jegfp The reaction condition of F/Jegfp R4 primer are as follows: 95 DEG C of 10min；95 DEG C of 50s, 57 DEG C of 30s, 72 DEG C of 100s, 35 circulations；72℃ 10min.PCR product carries out electrophoresis detection with 1% agarose.

The building of 1.14 PRV BJ- Δ gE/gI

In order to delete EGFP expression cassette, after pMD- Δ gE/gI-L-R is linearized with restriction enzyme EcoRI, With the PRV BJ- Δ gE/gI-EGFP DNA cotransfection ST cell extracted according to method in 1.10.After transfection, to ST cell When lesion reaches 70%~80%, harvest virus.Harvest virus carries out plaque purification, and picking does not express the plaque of green fluorescence. Plaque purification carries out 3 wheels, obtains the virus for being free of EGFP expression cassette, is named as PRV BJ- Δ gE/gI.

The identification of 1.15 PRVBJ- Δ gE/gI

PRV BJ- Δ gE/gI DNA is extracted, using PRV ID E/I up/PRV ID E/I down, PRV-gBf/PRV- GBr, PRV-gEf/PRV-gEr3 carry out PCR identification.PCR reaction system and reaction condition is the same as described in 1.13.

2 results

The transformation of 2.1 pEGFP-C1 plasmids

Plasmid pEGFP-C1 is subjected to double digestion with Bsp EI and BamH I and deletes extra restriction enzyme site, the matter being transformed Grain carries out double digestion identification with EcoR I and Ase I.Since EcoR I is deleted, improved plasmid pMEGFP-C1 is only by Ase I digestion goes out a band of a treaty 4.7kb, and it is about 1.3kb and two of about 3.4kb that the pEGFP-C1 plasmid not being transformed, which is digested, Band (Fig. 3).

The amplification of 2.2 or so homology arms

PRV Del E/I-L up/PRV Del E/I-L down and PRV Del E/I-R up/PRV Del is used respectively E/I-R down two carries out PCR to primer, by template of BJ plants of DNA of PRV, amplifies the left homology arm peace treaty of about 1200bp The right homology arm (Fig. 4) of 874bp.

The building of 2.3 pMD- Δ gE/gI-L-EGFP-R transfer vectors

The transfer vector pMD- Δ gE/gI-L-EGFP-R of building carries out double digestion mirror using EcoR I and Hind III Fixed, digestion goes out two bar segments and meets expection, and two segments (Fig. 5) of about 2.6kb and about 3.6kb, illustrate PRV gE/gI respectively Dual-gene missing transfer vector constructs successfully.

2.4 pMD- Δ gE/gI-L-EGFP-R transfer vector activity identifications

The transfer vector pMD- Δ gE/gI-L-EGFP-R of building is transfected in 293T cell.It is 24 hours to 48 small after transfection When, microscopically observation to cell expressing green fluorescent protein (Fig. 6).

2.5 knock out the building of the transfer vector pMD- Δ gE/gI-L-R of EGFP expression cassette

The pMD- Δ gE/gI-L-R metastasis transplanting physique grain of building carries out double digestion identification with EcoR I and Hind III, cuts Two DNA fragmentations of about 2.6kb and about 2kb out meet expected (Fig. 7).

The building of 2.6 PRV BJ- Δ gE/gI-EGFP

After BJ plants of DNA cotransfection ST cells of pMD- Δ gE/gI-L-EGFP-R transfer vector and PRV being linearized, 36~ There is within 48 hours cytopathy, the virus liquid of harvest obtains the PRV of expressing green fluorescent protein after 5 wheel plaque purifications BJ- Δ gE/gI-EGFP is viral (Fig. 8).

The building of 2.7 PRV BJ- Δ gE/gI

EGFP expression cassette, which is deleted, uses transfer vector, carries out double digestion through EcoR I and Xho I and cuts out about 5.9kb and about 2kb Two segments (Fig. 9), meet expected results, illustrate that the deletion of EGFP expression cassette is built into transfer vector PRV BJ- Δ gE/gI Function.

The acquisition of 2.8 PRV BJ- Δ gE/gI

By PRV BJ- Δ gE/gI-EGFP DNA and transfer vector PRV BJ- Δ gE/gI cotransfection is linearized to ST cell In, the gene delection virus PRV BJ- Δ gE/gI (Figure 10) of not expressing green fluorescent protein is filtered out by plaque purification. Fluorescence microscopy under the microscope delete successfully in PRV BJ- Δ gE/gI-EGFP genome as the result is shown by EGFP expression cassette.

The acquisition of 2.9 PRV BJ- Δ gE/gI

It is right using PRV ID E/I up/PRV ID E/I down, PRV-gBf/PRV-gBr, PRV-gEf/PRV-gEr 3 Primer pair PRV BJ- Δ gE/gI carries out PCR identification.Primer PRV ID E/I up/PRV ID E/I down is amplified about 1200bp purpose band；Primer PRV-gBf/PRV-gB amplifies 366bp purpose band；Primer PRV-gEf/PRV-gEr does not expand Increase purpose band out, PCR result meets expection, illustrates that PRV BJ- Δ gE/gI constructs successfully (Figure 11).

3 conclusions

With pseudo- mad BJ plants of dog variant for parent, the Pseudorabies virus BJ- Δ of the dual-gene missing of gE and gI is successfully constructed GE/gI plants, it is named as BJC plants.

Pathogenicity research of the dual-gene gene-deleted strain vaccine strain of 4 porcine pseudorabies virus gE/gI of embodiment (BJC plants) to piglet

1 materials and methods

1.1 viruses and cell

PRVBJC plants (preparation of embodiment 3)；ST cell is saved by this laboratory.

1.2 experimental animal

The susceptible piglet of 2~3 week old health, PCR method detection PRV antigen should be negative, and neutralize antibody titers≤1: 4, are purchased It is adapted at least 3 days after entering in animal house.

1.3 gE antibody assay kit IDEXX Products.

1.4 experimental method

1.4.1 inoculation and observation

Taking the susceptible piglet of 2~3 week old health, (PCR antigen is detected as feminine gender；Porcine pseudorabies serum neutralize antibody titers are not Higher than 1: 4) 10, it is randomly divided into 2 groups, every group 5, wherein the 1st group of every collunarium 3ml, intramuscular injection 3ml (10^7.0TCID₅₀/ ml)；2nd group is not inoculated with as control group.Isolated rearing.It is observed 14 after inoculation, record body temperature, feeding, spirit, morbidity or death Situation.

1.4.2gE antibody determination is carried out according to kit specification operation.

2 results

It is observed 14 after 2.1 inoculations, Pigs Inoculated feeding, spirit are normal, see Figure 12；Body temperature is normal, and compares pig indifference, Body temperature observation the results are shown in Table 10.

Body temperature observes table in 14 days after the inoculation of table 10

2.2 gE antibody test results

Inoculation group and the gE antibody test result of control group piglet are feminine gender, illustrate that the strain of building has lacked gE base Cause the results are shown in Table 11.

Inoculation groups on the 14th and control group gE antibody test result after table 11 is inoculated with

Note: "-" represents negative antibody.

3 conclusions

The present invention, which separates BJ plants of PRV obtained, can make 2~3 week old piglets morbidity or dead, pass through gene to BJ plants of PRV It knocks out and obtains PRVBJC plants (gE-gI- gene-deleted strains) 2~3 week old piglets of inoculation, body temperature, diet, normal, the gE of spirit in 14 days Antibody test is feminine gender, illustrates that the strain of the invention obtained eliminates the Disease-causing gene of parent, is one plant of less toxic power to piglet Strain.

The preparation of 5 BJC plants of inactivated vaccines of porcine pseudorabies virus of embodiment

1 seed culture of viruses

It is BJC plants of porcine pseudorabies virus that vaccine, which is manufactured, with seed culture of viruses, and inspection is BJ plants of porcine pseudorabies virus with seed culture of viruses.Nothing Bacterium, mould, mycoplasma and exogenous virus pollution.

The manufacture of 2 vaccines and the inspection of semifinished product

2.1 productions are prepared with seed culture of viruses

2.1.1 seed culture of viruses is bred

The ST cell for single layer of having grown up is discarded into growth-promoting media, seed culture of viruses BJC plants is inoculated with by 2% amount, is added and contains 2% new born bovine The DMEM cell culture fluid of serum, set 37 DEG C cultivate 2~3 days, the harvest when lesion occurs in 75% or more cell, set -15 DEG C with Lower freeze thawing 1 time, quantitative separating indicate harvest date, seed culture of viruses algebra (should be no more than for 5 generations) etc., -35 DEG C or less preservations, storage life It is 6 months.

The preparation of 2.2 cells

The breeding of 2.3 seedling venom

2.3.1 inoculation

2.3.2 harvest

2.3.3 viral level measures

2.4 inactivation

2.5 the inspection of semifinished product

2.5.1 inactivation is examined

2.5.2 steriling test

2.6 vaccine preparation

2.7 packing

4 product inspections

4.1 character appearance uniform emulsions.

The immunogenicity research of the dual-gene gene-deleted strain vaccine strain of 6 porcine pseudorabies virus gE/gI of embodiment (BJC plants)

1 materials and methods

1.1 vaccines with it is virulent

BJC plants of PRV (gE-gI- gene-deleted strain) inactivated vaccine (it is prepared by embodiment 4, and 10^7.5TCID50/ml), attack malicious with virulent BJ plants of PRV.

1.2 experimental animal

The susceptible piglet of 2~3 week old health, blood sampling, PCR method detection PRV antigen should be negative, neutralize antibody titers≤1: 4, it is adapted at least 3 days after buying in animal house.

1.3 gE antibody assay kits, gB antibody assay kit are purchased from IDEXX.

The test of 1.4 Immunizations

Susceptible piglet 25 of 2~3 week old health is taken, is randomly divided into 5 groups, every group 5, wherein the 5th group is not immunized controls Group, the 1st~4 group, PRV BJC strain vaccine 0.5ml, 1ml, 2ml and 3ml is immunized in musculi colli respectively, is immunized latter 21 days with same Dosage booster immunization is primary.21 days after booster immunization, together with control group, every collunarium 3ml, intramuscular injection 3ml (10^7.0TCID₅₀/ Ml) BJ plants it is virulent.It is observed 14 after attacking poison, record body temperature, feeding, spirit, morbidity or death condition.

1.5 gE antibody and gB antibody determination

It takes a blood sample before attacking poison to every immune swine, detects serum with gE antibody assay kit and gB antibody assay kit Antibody.

2 results

All piglet spirit are good after immune, and searching for food, it is normal to drink water, and the reaction of inoculation position no inflammation is immunized latter 24 hours occasionally There are individual body temperature to increase, but be less than 40.5 DEG C, compared with the control group no significant difference.After attacking poison, 0.5ml dosage group has 1 There is disease symptom, body temperature increases, and spirit is depressed；Remaining 4 there is transient body temperature and increase, without other clinical symptoms.1ml agent Amount group, 2ml dosage group and 3ml dosage group pig occur having no other clinical symptoms outside transient body temperature raising after attacking poison.Control Group attacks 5/5 morbidity after poison, and the 1st~2 day after attacking poison, all control temperature of pig body are increased to 41 DEG C or more, occur it is spiritual it is depressed, Subtract food and then anorexia, the nervous symptoms such as some appearance turn-take, dog seats, incoordination, finally dead 3.It attacks before poison to immune group GE antibody is not detected in all pigs, illustrates that the BJC strain of building does not generate gE antibody to piglet.All immune swines detect GB antibody illustrates that vaccine-induced pig produces immune response.Concrete outcome is shown in Table 12, and attacking poison after immune the results are shown in Table 13.

Immunoprotection result of the vaccine of the different immunizing doses of table 12 to piglet

3 conclusions

This result of study shows that BJC plants of porcine pseudorabies virus of minimum immune dosage (immunogenicity) is 10^7.5TCID₅₀/ 1ml is immunized in ml, for the immune effect for ensuring vaccine, recommended dose 2ml.

Claims

1. porcine pseudorabies virus velogen strain, which is characterized in that the porcine pseudorabies virus velogen strain is named as BJ, the poison Strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is in BeiChen West Road, Chaoyang District, BeiJing City No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute, culture presevation number are as follows: CGMCC No.15911, the deposit date is 2018 On July 11, in.

2. porcine pseudorabies virus velogen strain described in claim 1 is preparing the application in swine pseudorabies vaccine.

3. porcine pseudorabies virus velogen strain described in claim 1 establish porcine pseudorabies virus attack poison morbidity model in answering With.

4. according to claim 3 state application, which is characterized in that the porcine pseudorabies virus attacks poison morbidity model for epidemic disease In seedling efficacy test.

5. application according to claim 3 or 4, which is characterized in that use porcine pseudorabies virus described in claim 1 The pure culture of velogen strain carries out attacking poison.

6. porcine pseudorabies virus velogen strain described in claim 1 is preparing the dual-gene missing of porcine pseudorabies virus gE/gI Application in strain vaccine strain.

7. applying according to claim 6, which is characterized in that the dual-gene gene-deleted strain of porcine pseudorabies virus gE/gI Vaccine strain is to be obtained on the basis of the porcine pseudorabies virus velogen strain by the dual-gene building of knockout gE and gI.

8. a kind of swine pseudorabies vaccine, which is characterized in that contain the porcine pseudorabies described in claim 1 after inactivation Virus strain.

9. swine pseudorabies vaccine as claimed in claim 4, which is characterized in that take sterilizing 1 volume of ISA 206VG adjuvant Part, 1 parts by volume of porcine pseudorabies virus velogen strain virus liquid after inactivation is added, mixing is stirred at low speed 30~60 minutes, is made Vaccine.

10. swine pseudorabies vaccine as claimed in claim 8 or 9, which is characterized in that before inactivation, every milliliter of virus contains Amount answers >=10^7.5TCID50；The inactivation is will to examine qualified virus liquid, is slowly added to the two of final concentration of 0.05v/v% Aziridine (BEI) solution, it is stirring while adding, it is uniformly mixed, sets 30 DEG C and inactivate 48 hours, the thio sulphur of final concentration 2v/v% is added Acid sodium solution terminates inactivation.