CN106139139A - swine fever E2 subunit vaccine and application thereof - Google Patents
swine fever E2 subunit vaccine and application thereof Download PDFInfo
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- CN106139139A CN106139139A CN201510189303.7A CN201510189303A CN106139139A CN 106139139 A CN106139139 A CN 106139139A CN 201510189303 A CN201510189303 A CN 201510189303A CN 106139139 A CN106139139 A CN 106139139A
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- swine fever
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Abstract
The present invention provides a kind of swine fever E2 subunit vaccine and preparation method thereof, particularly as follows: utilize baculovirus vector expression system great expression restructuring swine fever E2 albumen in insect cell, develops the swine fever E2 subunit vaccine with good immune effect.Bac-to-Bac baculovirus expression system (Bac-to-Bac Baculovirus Expression System) is used to express CSFV E 2 protein.A kind of novel subunit vaccine is made in CSFV E 2 protein and adjuvant 201R emulsifying, by piglet immunological challenge test, verifies that this subunit vaccine has good immanoprotection action.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of swine fever E2 subunit vaccine and system thereof
Preparation Method.
Background technology
Swine fever (Classical Swine Fever, CSF), is by swine fever virus (Classical
Swine Fever Virus, CSFV) a kind of high degree in contact sexually transmitted disease of causing, pig is this disease
The unique natural reservoir (of bird flu viruses) of poison.It is near that it popular extends over the entire globe the most for over 100 years more than a hundred years.International Office of Epizootics is by it
It is set to A class infectious disease, is one of main epidemic disease endangering China's pig industry development at present.Epidemic disease
Seedling inoculation is the major measure of developing country's prevention and control swine fever.Current most widely used mainly
For swine fever attenuated vaccine, including China's hog cholera lapinised virus vaccine (C strain), Japan GPE-
Vaccine and the Thiverval vaccine of France.But, swine Fever Vaccine difficult quality guarantee in recent years
(mainly titre not and pollutes BVDV), often results in immuning failure.
Baculovirus expression system develops rapidly as a new research field.Outside substantial amounts of
Source gene obtains successful expression by rod string design.The spy of baculovirus
Levy the baculovirus particles being to be generally comprised within inclusion body (also becoming polyhedral body).Many albumen
Express in encoding the insect cell of recombinate shape virus infection of this protein.Shaft-like
Virus expression systems provides the powerful producing recombinant protein.The depletion of blood of insect cell
Clear culture technique is the most ripe, the Microcarrier Culture Techniques of insect cell and suspension culture
Technology provides condition for preparing recombinant protein on a large scale.China is every year because of swine fever virus
Outburst brings huge economic loss to China's pig industry, utilizes baculovirus expression system
In insect cell, great expression restructuring swine fever E2 albumen, develops and has good immune effect
Subunit vaccine, the anti-system for hog cholera viral disease has great importance.
CN103908663A discloses a kind of Novel pig Pestivirus E2 recombinant baculovirus inactivation
Vaccine and preparation method thereof, specifically discloses by the synthesis of E 2 gene of Classical Swine Fever, transfer
The structure of carrier, transfer vector and the restructuring of linearized baculovirus, the molecule of recombinant baculovirus
Biology and Immunological Identification, the expression of CSFV E 2 protein, swine fever virus E2 restructuring
The inactivation of baculovirus, CSFV E 2 protein make vaccine with adjuvant 563VG emulsifying.
Its prepared vaccine is to utilize the supernatant expressed to go direct immunization animal, and albumen does not pass through
Purification, has a lot of foreign protein and culture medium inside, and safety and effectiveness are substantially reduced.
Zhang Wenjie etc., 2007, Chinese experimental animal journal, 15 (1), swine fever virus E2 base
Because of the expression in Insect Cell/Baculovrius Expresion System, disclose E 2 gene of Classical Swine Fever thin insecticide
Expression in born of the same parents/baculovirus, specifically discloses: use RT-PCR to expand CSFV E2 base
Cause, is cloned into p-GEM-T-Easy carrier by PCR primer, is inserted into by this gene
In pFast-BacHT A carrier, build conversion DH10Bac competence after restructuring turns seat carrier thin
Born of the same parents, it is thus achieved that transfect sf9 insect cell after restructuring Bacmid plasmid, pass malicious 3 generations, to expression
Albumen carries out Western-blot and SABC is identified, successful clone CSFV raq gene,
SDS-PAGE electrophoresis result Explicit Expression E2 albumen relative molecular mass is about 43 × 103,
Western-blot and ImmunohistochemistryResults Results confirm that expressing protein can be by CSFV standard positive blood
Clear identification.
Summary of the invention
For solving the problems referred to above, it is an object of the invention to provide a kind of swine fever raq gene engineering
Subunit vaccine, its preparation method and application.
First, the present invention provides a kind of swine fever raq gene engineering subunit vaccine, and it contains anti-
Former and adjuvant, it is characterised in that described antigen is that baculovirus vector expression system is recombinant expressed
Swine fever E2 albumen, wherein, for the nucleotide sequence such as SEQ ID No.2 of swine fever E2 albumen
Shown in.
Wherein, described raq gene is synthetic, including: GP67 signal peptide nucleotide sequence
(129bp), E 2 gene of Classical Swine Fever partial sequence (1011bp), 6His sequence label
(18bp) and EcoR I restriction enzyme site (6bp) that holds of fragment 5 ' and fragment 3 ' are held
Xho I restriction enzyme site (6bp).
Wherein, described adjuvant is 201R VG water-in-oil-in water mineral oil adjuvant.
Subunit vaccine of the present invention, wherein, the consumption of every animal E2 albumen is
40-70μg。
The present invention also provides for the preparation method of swine fever raq gene engineering subunit vaccine, including such as
Lower step:
1) codon preference in insect cell, carries out the optimization of E 2 gene of Classical Swine Fever,
5 ' ends introduce GP67 signal peptide nucleotide sequence, the such as SEQ ID No.1 of the sequence after optimization
Shown in；
2) build containing in steps 1) transfer vector of swine fever raq gene, it is transformed into containing shuttling back and forth
In the competent escherichia coli cell of carrier, it is thus achieved that comprise the shuttle vector of swine fever raq gene,
Again this shuttle vector is transfected in insect cell, it is thus achieved that recombinant baculovirus；
3) recombinate shape virus infection insect cell and the cultivation of insect cell；
4) insect cell is expressed swine fever E2 recombiant protein, results and purification swine fever E2 albumen；
5) utilize swine fever E2 recombiant protein, be aided with adjuvant, through emulsifying, prepare swine fever E2 sub-
Subunit vaccine.
Wherein, adjuvant is mixed with antigen 1:1 in mass ratio, under the conditions of 200-400rpm,
By antigen and adjuvant homogenizing, the time is 2-10min, prepares swine fever E2 subunit vaccine.
The present invention also provides for described swine fever raq gene engineering subunit vaccine preparation preventing and treating pig
Purposes in the medicine of the disease that pestivirus infection causes.
Swine fever E2 albumen of the present invention is through His albumen after purification, effectively reduces miscellaneous
The side reaction that albumen causes；Swine fever E2 albumen Effective Antigens amount of the present invention is stable, it is possible to quantitatively,
Production easily determines between different batches the dosage of Effective Antigens.
The application swine fever E2 subunit vaccine antigenic purity prepared of technical scheme is high,
Safety is good, and immunogenicity is strong, and does not has pig pathogenic, for the purification of swine fever virus from now on
Material guarantee is provided.
Accompanying drawing explanation
Gene and vector construction schematic diagram for the purpose of Fig. 1
Fig. 2 is the PCR qualification figure of swine fever virus E2 recombinant baculovirus raq gene；
Fig. 3 is that recombinant baculovirus vBac-CSFV-E2 expresses at sf9 cell and Hi5 cell
Western-blot result；As shown in the figure: demonstrate recombinant baculovirus vBac-CSFV-E2
Expression in Hi5 cell is apparently higher than sf9 cell.
Fig. 4 is the ageing of the expression of CSFV E 2 protein and immunogenicity and expression
Western blot schemes.As shown in the figure: 1: albumen Marker；2,3,4,5,6,7 respectively
For E2 albumen after cultivation 24h, 36h, 48h, 60h, 72h and 96h；Demonstrate E2 egg
White 48h expression is the highest.
Fig. 5 is CSFV E 2 protein Histrap ni-sepharose purification result figure.
Fig. 6 is immune serum swine fever virus ELISA antibody test.As shown in the figure: card
Bright swine fever virus E2 recombinant subunit vaccine group antibody horizontal is apparently higher than swine fever E2 restructuring egg
The supernatant group of white insect cell.
Fig. 7 is that immune serum swine fever virus blocks ELISA antibody test.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
Prepared by embodiment 1 CSFV E 2 protein
1) E 2 gene of Classical Swine Fever optimization and synthesis
With reference to domestic and international classical strains and the raq gene of epidemic isolates, Select gene hypotype 2.1
Swine fever virus (gene order is Classical swine fever virus strain Zj0801,
Complete genome GenBank:FJ529205.1) press the inclined preferendum of codon in insect cell
Carry out the optimization of E 2 gene of Classical Swine Fever, give company to synthesize, the gene bag of synthetic
Include: GP67 signal peptide nucleotide sequence (129bp), E 2 gene of Classical Swine Fever partial sequence
(1011bp) the EcoR I enzyme action position that, 6His sequence label (18bp) and fragment 5 ' are held
Xho I restriction enzyme site (6bp) (see Fig. 1) (the SEQ ID No.2 that point (6bp) and fragment 3 ' are held
Shown in).
2) clone of gene:
The exogenous dna fragment of synthetic is connected to after EcoR I and Xho I double digestion
PFastBac1 plasmid, cloned plasmids method for determining nucleic acid sequence is verified.Enzyme action, company
Connect, conversion etc. is conventional molecular biological method.
3) pFastBac CSFV E2 is converted the escherichia coli containing shuttle plasmid Bacmid
DH10Bac (purchased from Invitrogen company), it is thus achieved that recombiant plasmid Bacmid-CSFV-E2；
PCR screens, and extracts positive plasmid, the positive recombiant plasmid of preparation
Bacmid-CSFV-E2DNA.UseII Reagent transfection SF9 cell (is purchased
From Invitrogen company), after there is pathological changes in cell, carry out recombinant virus plaque purification and
Virus amplification, then carries out PCR by universal primer M13F and M13R and verifies purpose weight
Group raq gene (see Fig. 2).Purification obtains recombinant baculovirus, named
vBac-CSFV-E2.Recombinant baculovirus vBac-CSFV-E2 is as kind of a poison in amplification, passes through
Plaque ethods saves backup in 4 DEG C after carrying out titer titration.
4) recombinant baculovirus vBac-CSFV-E2 infects sf9 cell and High Five respectively
Cell and cultivation, optimum combination baculovirus vBac-CSFV-E2 is in different insects cell
Expression.Result shows that recombinant baculovirus vBac-CSFV-E2 is at High Five cell
In expression all may be used apparently higher than sf9 cell (see Fig. 3), sf9 and High Five cell
For expressing, more preferable with High Five during expression-secretion albumen.
5) the insect cell High of vBac-CSFV-E2 recombinate shape virus infection suspension culture
Five
In the shaking flask of the 1L of the culture medium containing 400ml, sterile suspension cultivates High Five
Cell, inoculating cell density is 0.35～0.6 × 106Individual cell/ml.When cell density reaches
1.8～2.5 × 106During individual cell/ml, inoculate shaking flask with recombinant virus vBac-CSFV-E2, connect
Infection multiplicity (MOI) suggestion of the recombinant baculovirus planted in each shaking flask is 0.5-1.5, connects
After kind of recombinant baculovirus, with 27 DEG C, rotating speed is 120rpm, respectively at the 24h cultivated,
Sampling after 36h, 48h, 60h, 72h and 96h, western blot verifies swine fever virus E2 egg
White expression and immunogenicity and ageing (see the Fig. 4) of expression.Result shows 48h swine fever
E2 expressing quantity is the highest, pours in centrifuge bottle centrifugal after 48h after being shaken up by Cell sap, with
Rotating speed 9000rpm is centrifuged 1h, collects supernatant and is CSFV E 2 protein liquid.
6) the swine fever raq gene engineering subunit vaccine preparation purification of E2 albumen
Collecting albumen: 4 DEG C, 9000rpm is centrifuged 1h, abandon precipitation, supernatant is filtered by 0.22 μm
Film sucking filtration, with remove impurity prevent block Histrap.
Histrap nickel post: first use deionized water the NiSO in pillar before use4Wash off, then
20mM Tris, 150mM NaCl, pH8.0 is used to balance.The good albumen supernatant of sucking filtration is with compacted
Dynamic pump overnight combines Histrap at freezer (4 DEG C), calculates the time, and calculates with Ep pipe
Good flow velocity, is sure not to drain.After finished white supernatant, Histrap is unloaded, is installed to
On AKTA, flow velocity 2.5ml/min (AKTA first uses 20mM Tris, 150mM NaCl,
PH8.0 washes pump, UV value zeroing after walking to put down), use 20mM Tris, 150mM NaCl,
PH8.0 Buffer washes foreign protein, rushes shallower to UV value, then uses 20mM imidazoles,
The foreign protein of 20Mm Tris, 150mM NaCl, pH8.0 eluting non-specific binding, punching is extremely
UV value is shallower, finally uses 300mM imidazoles, 20mM Tris, 150mM NaCl,
PH8.0 eluting destination protein, punching to UV value is shallower, and (typically collecting 200mAU is
Only).Albumin glue (see Fig. 5) is run in sampling.Destination protein is collected and is concentrated to about 20mL,
Freezer is dialysed, uses 20mM Tris, 150mM NaCl, pH8.0 Buffer to dialyse, thoroughly
Analysis liquid is long-pending should be albumen volume more than 20 times, changes liquid once after 4 hours.
Prepared by embodiment 2 CSFV E 2 protein subunit vaccine
Vaccine is made in CSFV E 2 protein and 201R emulsifying: the preparation of adjuvant, 201R helps
Agent (121 DEG C, 15pa, 20min) carries out sterilizing, is mixed with adjuvant 1:1 by proteantigen,
With rotating speed 350rpm emulsifying 5min, i.e. prepare swine fever E2 recombinant subunit vaccine.
Test example 1
With 30 BALB/c 5-6 week old female mices, it is divided into 3 groups, often group 10,1
Group is swine fever virus E2 recombinant subunit vaccine group (12.5 μ g/ head part), and two groups is swine fever E2
The supernatant group (12.5 μ g/ head part) of recombiant protein insect cell is (by CN103908663A system
Standby), 3 groups is placebo group, respectively at mice femoribus internus intramuscular injection vaccine, during injection
Between be 0d, 21d, blood sampling time is 0d, 21d, 35d.Utilize indirect ELISA detection anti-
Body titre (see Fig. 6), result shows the immunity effect of swine fever virus E2 recombinant subunit vaccine
Fruit is apparently higher than the immune effect of the supernatant of swine fever E2 recombiant protein insect cell.
The efficacy test of the swine fever virus E2 recombinant subunit vaccine of the present invention: with 30 4～
(swine fever virus antigen-antibody is feminine gender, immunosuppressive disease to the healthy susceptible piglet of 5 week old
(reproductive and respiratory syndrome, circovirus disease) antigen negative pig), it is divided into 6 groups, often group 5,1-4
Group respectively swine fever virus E2 recombinant subunit vaccine various dose group 50 μ g/ head part,
200 μ g/ head parts, 500 μ g/ head parts, 1000 μ g/ head parts, the 5th group of immunity swine fever spleen drenches Seedling,
6th group as a control group, under equal conditions isolated rearing, and one exempts from rear 21d with same dose
Carrying out two with approach to exempt from, two exempt from rear 21d, all pig musculi collis injections swine fever crossdrift system blood poison
1ml(106TCID50/ ml) carry out counteracting toxic substances, monitor pig body temperature 14d, Pigs Inoculated afterwards every day
Pestilence E2 recombinant subunit vaccine and swine fever spleen drench 5 groups of test pig body temperature of Seedling and have no rising, cut open
Finding after inspection, tonsil, larynx, lymph node, kidney, spleen, ileocecal valve are showed no out
Blood point and other abnormal pathologic situations, after matched group test pig counteracting toxic substances, body temperature raise 40 DEG C with
On, to become thin, diarrhoea, ataxia, after counteracting toxic substances, 5d and 12d dead 1 respectively, cuts open inspection
Time tonsil, larynx, lymph node, kidney, spleen, ileocecal valve all have petechia or hemorrhage
Speckle, splenic marginal infarction, lymph node tangent plane is marble sample decorative pattern, and kidney surface has in a large number
Petechia, presents typical case's " Passeris montani saturati kidney " pathological change.Application blocks ELISA and detects antibody
Titre, result is shown in that Fig. 7, result show that swine fever E2 subunit vaccine immune swine is after immunity
3w, swine fever specificity neutralizing antibody starts to produce, and after booster immunization, antibody persistently raises, and
Within 3 weeks after booster immunization, reaching peak value, before being continued until counteracting toxic substances, after counteracting toxic substances, antibody is rapid
Raise, and be continued until that experiment terminates, and matched group placebo group immune swine does not all produce
Swine fever specific antibody.
The above is only the preferred embodiment of the present invention, it is noted that lead for this technology
For the those of ordinary skill in territory, on the premise of without departing from the technology of the present invention principle, it is also possible to
Making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (6)
1. a swine fever raq gene engineering subunit vaccine, it contains antigen and adjuvant, and it is special
Levying and be, described antigen is the swine fever E2 albumen that baculovirus vector expression system is recombinant expressed,
Wherein, for expressing the nucleotide sequence of swine fever E2 albumen as shown in SEQ ID No.2.
2. subunit vaccine as claimed in claim 1, it is characterised in that described adjuvant is
201R VG water-in-oil-in water mineral oil adjuvant.
3. subunit vaccine as claimed in claim 1 or 2, it is characterised in that every is moved
The consumption of thing E2 albumen is 40-70 μ g.
4. the system of the swine fever raq gene engineering subunit vaccine described in any one of claim 1-3
Preparation Method, comprises the steps:
1) codon preference in insect cell, carries out the optimization of E 2 gene of Classical Swine Fever,
5 ' ends introduce GP67 signal peptide nucleotide sequence, the such as SEQ ID No.1 of the sequence after optimization
Shown in；
2) build containing in steps 1) transfer vector of swine fever raq gene, it is transformed into containing shuttling back and forth
In the competent escherichia coli cell of carrier, it is thus achieved that comprise the shuttle vector of swine fever raq gene,
Again this shuttle vector is transfected in insect cell, it is thus achieved that recombinant baculovirus；
3) recombinate shape virus infection insect cell and the cultivation of insect cell；
4) insect cell is expressed swine fever E2 recombiant protein, results and purification swine fever E2 albumen；
5) utilize swine fever E2 recombiant protein, be aided with adjuvant, through emulsifying, prepare swine fever E2 sub-
Subunit vaccine.
5. method as claimed in claim 4, it is characterised in that by adjuvant with antigen by matter
Measuring and mix than 1:1, under the conditions of 200-400rpm, by antigen and adjuvant homogenizing, the time is
2-10min, prepares swine fever E2 subunit vaccine.
6. the swine fever raq gene engineering subunit vaccine described in any one of claim 1-3 is in system
Purposes in the medicine of the disease that standby preventing and treating swine fever virus infection causes.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106110319A (en) * | 2016-07-04 | 2016-11-16 | 中国兽医药品监察所 | E 2 gene of Classical Swine Fever recombinant baculovirus inactivated vaccine preparation method |
| CN108395996A (en) * | 2018-01-31 | 2018-08-14 | 复旦大学 | A kind of swine fever virus subunit vaccine and its preparation method and application |
| CN108845149A (en) * | 2018-07-05 | 2018-11-20 | 郑州大学 | The colloidal gold duplex test strips and preparation method thereof of porcine reproductive and respiratory syndrome virus and swine fever virus are detected simultaneously |
| CN110029116A (en) * | 2019-03-12 | 2019-07-19 | 华南农业大学 | The recombinant virus and preparation method of a kind of more epitope E 2 gene of Classical Swine Fever of secreting, expressing and application |
| CN110041411A (en) * | 2018-01-15 | 2019-07-23 | 浙江海隆生物科技有限公司 | Stable atypical classical swine fever virus subunit protein, vaccine thereof, preparation method and application |
| CN110747215A (en) * | 2019-11-01 | 2020-02-04 | 北京鼎持生物技术有限公司 | Recombinant baculovirus for efficiently expressing hog cholera E2 protein and construction method thereof |
| CN110950968A (en) * | 2019-12-17 | 2020-04-03 | 天康生物(上海)有限公司 | gp 96-hog cholera E2 fusion protein, preparation method thereof and vaccine |
| CN111973738A (en) * | 2020-09-02 | 2020-11-24 | 天康生物股份有限公司 | Nucleic acid and recombinant protein co-immune vaccine based on hog cholera virus gene, preparation method and application |
| CN113384691A (en) * | 2021-06-11 | 2021-09-14 | 湖南兀邦生物科技有限公司 | Classical swine fever virus E2 protein recombinant subunit vaccine taking salmonella flagellin as molecular adjuvant and preparation method thereof |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106110319A (en) * | 2016-07-04 | 2016-11-16 | 中国兽医药品监察所 | E 2 gene of Classical Swine Fever recombinant baculovirus inactivated vaccine preparation method |
| CN106110319B (en) * | 2016-07-04 | 2019-12-20 | 中国兽医药品监察所 | Preparation method of classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine |
| CN110041411A (en) * | 2018-01-15 | 2019-07-23 | 浙江海隆生物科技有限公司 | Stable atypical classical swine fever virus subunit protein, vaccine thereof, preparation method and application |
| CN110041411B (en) * | 2018-01-15 | 2023-10-03 | 浙江海隆生物科技有限公司 | Stable atypical swine fever virus subunit protein, vaccine, preparation method and application thereof |
| CN108395996B (en) * | 2018-01-31 | 2021-07-23 | 复旦大学 | Classical swine fever virus subunit vaccine and preparation method and application thereof |
| CN108395996A (en) * | 2018-01-31 | 2018-08-14 | 复旦大学 | A kind of swine fever virus subunit vaccine and its preparation method and application |
| CN108845149A (en) * | 2018-07-05 | 2018-11-20 | 郑州大学 | The colloidal gold duplex test strips and preparation method thereof of porcine reproductive and respiratory syndrome virus and swine fever virus are detected simultaneously |
| CN110029116A (en) * | 2019-03-12 | 2019-07-19 | 华南农业大学 | The recombinant virus and preparation method of a kind of more epitope E 2 gene of Classical Swine Fever of secreting, expressing and application |
| CN110747215A (en) * | 2019-11-01 | 2020-02-04 | 北京鼎持生物技术有限公司 | Recombinant baculovirus for efficiently expressing hog cholera E2 protein and construction method thereof |
| CN110950968A (en) * | 2019-12-17 | 2020-04-03 | 天康生物(上海)有限公司 | gp 96-hog cholera E2 fusion protein, preparation method thereof and vaccine |
| CN110950968B (en) * | 2019-12-17 | 2021-08-17 | 天康制药(苏州)有限公司 | gp 96-hog cholera E2 fusion protein, preparation method thereof and vaccine |
| CN111973738A (en) * | 2020-09-02 | 2020-11-24 | 天康生物股份有限公司 | Nucleic acid and recombinant protein co-immune vaccine based on hog cholera virus gene, preparation method and application |
| CN113384691A (en) * | 2021-06-11 | 2021-09-14 | 湖南兀邦生物科技有限公司 | Classical swine fever virus E2 protein recombinant subunit vaccine taking salmonella flagellin as molecular adjuvant and preparation method thereof |
| CN113384691B (en) * | 2021-06-11 | 2022-08-16 | 湖南兀邦生物科技有限公司 | Classical swine fever virus E2 protein recombinant subunit vaccine taking salmonella flagellin as molecular adjuvant and preparation method thereof |
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