CN105924506A - Preparation method of bovine viral diarrhea virus E2 protein subunit vaccine - Google Patents
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Abstract
The invention relates to a preparation method of a bovine viral diarrhea virus E2 protein subunit vaccine. The preparation method has the following advantages that: firstly, an expression method for obtaining BVDV E2 protein efficiently is set; and secondly, the E2 protein of the BVDV is used for expressing a supernatant immune rabbit, is proved to have good immunoreactions, and can be used for preparing BVDV subunit vaccine.
Description
Technical field
Bovine viral diarrhea virus (BVDV) E2 protein expression involved in the present invention and subunit vaccine preparation method thereof,
Belong to veterinary biologics field.
Background technology
The diarrhoea of the cattle that bovine viral diarrhea virus (bovine viral diarrhea virus, BVDV) can cause and reproductive failure.
After Pregnant cows infects BVDV, stillbirth, miscarriage and embryo's deformity can be caused, or give a birth out persistent infection calf.This
Though the calf of kind of persistent infection does not show any clinical symptoms, but can band poison all the life, persistently toxin expelling, becomes the important source of infection,
It is widely present in China, causes great economic loss to China's cattle-raising.
BVDV belongs to flaviviridae pestivirus, and its genome is single-stranded positive RNA, and total length about 12.5Kb contains only
One can encode about 4000 ORF that aminoacid polyprotein is big, polyprotein translated and processing after, 11 kinds can be formed
Ripe albumen, wherein C, Erns, E1, E2 are the structural protein of virus.E2 is the envelope protein of BVDV, immunity
Originality is the strongest, body can be induced simultaneously to produce humoral immunization and cellular immunization, and produce neutralizing antibody, be to prepare detection antigen
With the preferred genes of vaccine, BVDV E2 is expressed and immunogenic research has been carried out a lot.Marzocca MP
Expressing in Drosophila melanogaster Deng by raq gene, the E2 protein expression of restructuring goes out good antigenicity (Truncated E2of
bovine viral diarrhea virus(BVDV)expressed in Drosophila melanogaster cells:a candidate
antigen for a BVDV ELISA.J Virol Methods,2007,144(1-2):49-56)；Ferrer F etc. uses Rachiplusia
Nuperos baculovirus protein expression system obtains E2 recombiant protein, the experiment proved that, this albumen can induce neutralizing antibody
Produce, can be used for preparing vaccine antigen (Induction ofvirus neutralizing antibodies by immunization with
Rachiplusia nuperos infected with a recombinant baculovirus expressing the E2glycoprotein of
Bovine viral diarrhea virus.J Virol Methods, 2007,146 (1-2): 424-427).Domestic have employed many
Kind of mode to its express (such as: Xu Xing so etc., bovine viral diarrhea virus Changchun184 strain raq gene gram
Grand and high efficient expression in large intestine bar), but prokaryotic expression product is inclusion body, without correct structure, poor activity.Therefore,
The expression of E2 albumen need to be studied further, to obtain solvable highly active protein.
Research finds, different plant species has the biggest difference to the use frequency of amino acid codes, therefore, by BVDV in the present invention
Raq gene is codon optimized for insect baculovirus preferred codons, to improve the expression of BVDV E2.
It is an object of the invention to set up BVDV E2 albumen and subunit vaccine preparation method thereof.
Summary of the invention
Technical scheme
1. bovine viral diarrhea virus E2 protein subunit vaccine preparation method, it is characterised in that this vaccine is by building
It is named as autogra-phacalifornica nuclear polyhedrosis virus (being called for short baculovirus) BacVDMoptiE2 strain as producing strain
Being prepared from, this strain delivered the Chinese section in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on 06 01st, 2016
Institute of microbiology of institute China Microbiological preservation committee common micro-organisms preservation center, preserving number is respectively as follows: CGMCC
No.12545。
2. bovine viral diarrhea virus E2 protein subunit vaccine preparation method described in claim 1, it is characterised in that its structure
The baculovirus BacVDMoptiE2 strain built is carried MoptiE2-1 (sequence 1) and MoptiE2-2 (sequence 2).
3. bovine viral diarrhea virus (BVDV) E2 protein subunit vaccine preparation method as claimed in claim 1, it is special
Levy and be to carry out expressing with its baculovirus BacVDMoptiE2 strain built to obtain soluble E 2 albumen, through inactivation, add
It is prepared as subunit vaccine after entering suitable adjuvant emulsifying.
The main technical principle of the present invention
With reference to BVDV raq gene sequence, use the means of full genome synthesis, the BVDV raq gene sequence of synthesis optimizing
(optiE2)；Again by overlap extension pcr respectively by BVDV raq gene sequence (optiE2) optimized and secretion
Signal peptide Mels gene fusion, it is thus achieved that transfer vector；
Transfer vector is cloned in Baculovirus Gene group by transposition technology, builds recombinant baculovirus, it is thus achieved that BVDV
E2 albumen；
The BVDV E2 albumen obtained is added suitable adjuvant, immune animal after emulsifying, analyzes it as subunit vaccine
Feasibility；
The specific embodiment of the invention
The acquisition of 1.BVDV raq gene sequence
Use the means of full genome synthesis, BVDV raq gene sequence (optiE2) of synthesis optimizing, be cloned into pMD 18
Carrier, named: plasmid poptiE2.
2.E2 gene merges with signal peptide sequence
Extracting poptiE2, design primer also adds suitable restriction enzyme site, by overlap extension pcr (Overlap extension PCR
Technology and the application [J] in genetic engineering thereof. Molecular Plant Breeding, 2006,05:747-750) obtain and signal peptide Mels gene
Merge, be cloned into pMD 18 carrier.The fusion gene plasmid built is respectively designated as pMoptiE2-1, pMoptiE2-2.
The sequence carried in fusion gene plasmid pMoptiE2-1, it may be assumed that MoptiE2-1 (sequence 1):
BamHI restriction enzyme site is added in upstream, and termination codon and HindIII site are added in downstream
The sequence carried in fusion gene plasmid pMoptiE2-2, it may be assumed that MoptiE2-2 (sequence 2):
SmaI restriction enzyme site is added in upstream, and termination codon and KpnI site are added in downstream
3. sub-clone is to baculovirus transfer vector
PMoptiE2-1 and carrier pFastBacDual carries out double digestion respectively, and purification reclaims, enzyme action segment MoptiE2-1
Be connected with pFastBacDual linear plasmid, convert, identify, it is thus achieved that the named pFB-MoptiE2 of novel plasmid；Again will
PMoptiE2-2 and carrier pFB-MoptiE2 carries out double digestion respectively, and purification reclaims, enzyme action segment MoptiE2-2 with
PFB-MoptiE2 linear plasmid connects, and converts, and identifies, it is thus achieved that the named pFB-DMoptiE2 of novel plasmid (D represents double
Express).
4. swivel base obtains restructuring bacmid dna
Respectively the bacmid dna that pFB-MoptiE2, pFB-DMoptiE2 conversion DH10Bac constructs is respectively designated as:
bMoptiE2、bDMoptiE2。
5. the acquisition of recombinant virus
Respectively restructuring rod granule bMoptiE2, bDMoptiE2 transfection reagent Cellfectin is transfected sf9 cell, expand training
Support, collect culture supernatant and carry out PCR qualification.Obtain recombinant baculovirus named baculovirus BacVMoptiE2 respectively
Strain and baculovirus BacVDMoptiE2, the Classification And Nomenclature of this two strain suggestion is autogra-phacalifornica nucleopolyhedrosis
Poison, wherein baculovirus BacVDMoptiE2 delivered BeiChen West Road, Chaoyang District, BeiJing City 1 on 06 01st, 2016
Number No. 3 China Microbiological preservation committee of Institute of Microorganism, Academia Sinica common micro-organisms preservation centers of institute, preserving number divides
It is not: CGMCC No.12545.
6. expression analysis
The viral passages amplification culture that will have identified, the second filial generation is designated as P2Stock, and the third generation is designated as P3Stock.First three generation
All as kind of a poison, take the sf9 cell that the 3rd culture supernatant is inoculated in 48 orifice plates, set simultaneously and do not connect poison comparison.After 60 hours
Abandon and ask, fix with the fixative (acetone: methanol=1:1) of pre-cooling.Abandon fixative, add BVDV McAb (from AHVLA),
Hatch 50min for 37 DEG C, wash 3 times with PBS, add the sheep anti-mouse antibody of FITC labelling, hatch 50min, use PBS for 37 DEG C
Wash 3 times, fluorescence microscopy Microscopic observation.Result shows, connects poison sf9 cell hole and obvious bright green fluorescence occurs (in Fig. 1
Figure A, schemes B), and do not connect poison sf9 cell hole and do not show fluorescence (the figure E in Fig. 1).Illustrate that recombinant baculovirus can be expressed
BVDV E2 albumen.
7. Seedling is joined in emulsifying
As a example by BacVDMoptiE2 expressing protein joins Seedling.
To identify that correct kind poison sf9 cell amplification, to P6 generation, measures virus titer.Use ExpressFive culture medium
(Invitrogen company) suspension culture High Five insect cell (Invitrogen company) (27 DEG C, 180r/min shakes cultivation),
When High Five insect cell length is to exponential phase, the ExpressFive culture medium more renewed, adjusting cell concentration is
2～5 × 10-6/ ml, by recombinant baculovirus BacVDMoptiE2 with 1MOI dosage infection cell, gathers in the crops after 96 hours.
2000r/min is centrifuged 10min, takes supernatant, carries out SDS-PAGE electrophoresis, calculates target product content.BEI is added
In antigen, final concentration of lmmol/L, puts 37 DEG C of inactivation 48h, can be inactivated by recombinant virus.
The preparation of 8.BVDV subunit vaccine and animal immune
As a example by BacVDMoptiE2, BacVDMoptiE2 is expressed after supernatant concentrates 10 times and add in 1:1 (v/v) ratio
Entering ISA206 adjuvant, after emulsifying, immunity large ear rabbit, subcutaneous multi-point injection, every 1ml, add after 28 days and exempt from once.Add
Take a blood sample after exempting from 14 days, separate serum, 56 DEG C of inactivation 30min.Neutralization test is done with the serum separated.Method: dilution BVDV
OregonC24 to 300TCID50/ ml, by serum with MEM by 2 ×, 4 ×, 8 ×, 16 ×, 32 ×, 64 ×, 128 ×, 256 ×
Dilution, 37 DEG C of effect 60min, then it is inoculated in ready 48 hole MDBK cell plates, 37 DEG C of absorption 90min.
Then blotting, every hole adds the MEM culture fluid 0.5ml, 37 DEG C of CO containing 2%FBS2Incubator cultivates 72h.Blot,
Rinse 3 times with PBS, blot, dye with BVDV fluorescent antibody liquid.During the hole having typical green florescent signal represents not
With.
Result shows, with titer up to 1:128 in serum, can induce generation after BVDV subunit vaccine immune rabbit is described
Neutralizing antibody, can use as candidate vaccine.
The microbial resources information that the present invention relates to
The present invention builds the recombinant baculovirus BacVDMoptiE2 strain of acquisition, and male parent is from DH10Bac bacterium
Baculovirus Gene group in (Invitrogen company), by by exogenous gene BVDV raq gene and secreting signal peptide
After the fusion gene complete DNA cloning of Mels enters 2 reading frames of pFastBacDual plasmid, shaft-like with DH10Bac bacterium
The new baculovirus BacVDMoptiE2 strain obtained after viral genome restructuring, the Classification And Nomenclature of this strain suggestion is Herba Medicaginis
Silver mosquito californica nuclear polyhedrosis virus, and delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on 06 01st, 2016
Number China Microbiological preservation committee of Institute of Microorganism, Academia Sinica common micro-organisms preservation center, preserving number is respectively as follows:
CGMCC No.12545。
Accompanying drawing explanation
Fig. 1 wherein schemes A-D and shows that connecing poison sf9 cell hole obvious bright green fluorescence occurs, and figure A. is BacVME2, figure
B. being BacVDME2, figure C. is BacVMoptiE2, and figure D. is BacVDMoptiE2；E. for not connecing poison sf9 cell
Comparison do not show fluorescence
The technology path of Fig. 2 present invention
Advantages of the present invention
The present invention relates to bovine viral diarrhea virus E2 protein subunit vaccine preparation method.Its advantage is as follows: 1. establish
The expression of effective acquisition BVDV E2 albumen；2. with the E2 protein expression supernatant immune rabbit of BVDV, it was demonstrated that have
Good immunoreation, can be used for preparing BVDV subunit vaccine.
Embodiment
Embodiment 1
The structure of recombinant baculovirus
According to the principle that the present invention is same, construct the recombinant baculovirus containing natural B VDV raq gene the most simultaneously.
The acquisition of 1.BVDV raq gene sequence
BVDV BA strain (CVCC AV69) infected material extracts viral RNA, by conventional RT-PCR technology, obtains
Obtain BVDV raq gene, be cloned into pMD 18 carrier, named: plasmid pE2.
2.E2 gene merges with signal peptide sequence
Extracting pE2, design primer also adds suitable restriction enzyme site, is obtained and signal peptide Mels by overlap extension pcr
Gene fusion, is cloned into pMD 18 carrier.The fusion gene plasmid built is respectively designated as pME2-1, pME2-2.
The sequence carried in fusion gene plasmid pME2-1, it may be assumed that ME2-1 (sequence 3):
BamHI restriction enzyme site is added in upstream, and termination codon and HindIII site are added in downstream
The sequence carried in fusion gene plasmid pME2-2, it may be assumed that ME2-2 (sequence 4):
SmaI restriction enzyme site is added in upstream, and termination codon and KpnI site are added in downstream
3. sub-clone is to baculovirus transfer vector
PME2-1 and carrier pFastBacDual carries out double digestion respectively, and purification reclaims, enzyme action segment ME2-1 with
PFastBacDual linear plasmid connect, convert, identify, it is thus achieved that the named pFB-ME2 of novel plasmid；Again by pME2-2
Carry out double digestion respectively with carrier pFB-ME2, purification reclaims, and enzyme action segment ME2-2 is connected with pFB-ME2 linear plasmid,
Convert, identify, it is thus achieved that the named pFB-DME2 of novel plasmid (D represents double expression(DE))；
4. swivel base obtains restructuring bacmid dna
Respectively the bacmid dna that pFB-ME2, pFB-DME2 conversion DH10Bac constructs is respectively designated as: bME2,
bDME2。
5. the acquisition of recombinant virus
Respectively restructuring rod granule bME2, bDME2 transfection reagent Cellfectin is transfected sf9 cell, amplification culture, collect
Culture supernatant carries out PCR qualification.Respectively obtain recombinant baculovirus named baculovirus BacVME2 strain and
BacVDME2 strain, the Classification And Nomenclature of this two strain suggestion is autogra-phacalifornica nuclear polyhedrosis virus.
Embodiment 2
Expression of recombinant virus is analyzed
The viral passages amplification culture that will have identified, the second filial generation is designated as P2Stock, and the third generation is designated as P3Stock.First three generation
All as kind of a poison, take the sf9 cell that the 3rd culture supernatant is inoculated in 48 orifice plates, set simultaneously and do not connect poison comparison.After 60 hours
Abandon and ask, fix with the fixative (acetone: methanol=1:1) of pre-cooling.Abandon fixative, add BVDV McAb (from AHVLA),
Hatch 50min for 37 DEG C, wash 3 times with PBS, add the sheep anti-mouse antibody of FITC labelling, hatch 50min, use PBS for 37 DEG C
Wash 3 times, fluorescence microscopy Microscopic observation.Result shows, connects poison sf9 cell hole and obvious bright green fluorescence occurs (in Fig. 1
Figure C and figure D), and do not connect poison sf9 cell hole and do not show fluorescence (the figure E in Fig. 1).Illustrate that recombinant baculovirus can be expressed
BVDV E2 albumen.
Result shows, with titer up to 1:128 in serum, can induce generation after BVDV subunit vaccine immune rabbit is described
Neutralizing antibody, can use as candidate vaccine.
Claims (3)
1. bovine viral diarrhea virus E2 protein subunit vaccine preparation method, it is characterised in that this vaccine is to be named as by build
Autogra-phacalifornica nuclear polyhedrosis virus (being called for short baculovirus) BacVDMoptiE2 strain is prepared from as producing strain, this strain
The micro-life of No. 3 China of Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City has been delivered on 06 01st, 2016
Thing preservation committee's common micro-organisms preservation center, preserving number is respectively as follows: CGMCC No.12545.
2. bovine viral diarrhea virus E2 protein subunit vaccine preparation method described in claim 1, it is characterised in that it is shaft-like that it builds
MoptiE2-1 (sequence 1) and MoptiE2-2 (sequence 2) is carried in virus BacVDMoptiE2 strain.
3. as claimed in claim 1 bovine viral diarrhea virus E2 protein subunit vaccine preparation method, it is characterised in that build with it
Baculovirus BacVDMoptiE2 strain carries out expressing acquisition soluble E 2 albumen, through inactivation, is prepared as Asia after adding suitable adjuvant emulsifying
Subunit vaccine.
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| CN111748584A (en) * | 2020-05-28 | 2020-10-09 | 东北农业大学 | Wide-host expression vector, recombinant BacMam virus prepared based on vector and application of recombinant BacMam virus |
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| CN108641993A (en) * | 2018-05-17 | 2018-10-12 | 新疆农垦科学院 | A kind of recombinant lactic acid bacteria oral vaccine strain of food-grade expression bovine viral diarrhea virus E2 albumen |
| CN110004178A (en) * | 2019-04-02 | 2019-07-12 | 石河子大学 | A kind of preparation method of the preparation of bovine viral diarrhea virus sample particle |
| CN111748584A (en) * | 2020-05-28 | 2020-10-09 | 东北农业大学 | Wide-host expression vector, recombinant BacMam virus prepared based on vector and application of recombinant BacMam virus |
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