CN108059684A - A kind of bovine viral diarrhoea recombinant protein, its preparation method and application - Google Patents
A kind of bovine viral diarrhoea recombinant protein, its preparation method and application Download PDFInfo
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- bovine viral
- viral diarrhoea
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- 206010051511 Viral diarrhoea Diseases 0.000 title claims abstract description 79
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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Abstract
The invention discloses a kind of bovine viral diarrhoea recombinant protein, including bovine viral diarrhoea antigen and bovine viral diarrhoea single-chain antibody：Bovine viral diarrhoea antigen is the E2 antigens containing major antigenic determinant；Bovine viral diarrhoea single-chain antibody is scFv.The invention also discloses the preparation method and applications of the bovine viral diarrhoea recombinant protein.The present invention is prepared for combining the recombinant protein of the high activity of bovine viral diarrhoea antibody and antigen, high sensitivity and high specific, and the cross reaction between raw material is avoided to reduce the input of production cost simultaneously.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to a kind of bovine viral diarrhoea recombinant protein, its preparation method and
Using.
Background technology
Bovine viral diarrhoea (Bovine viral diarrhea virus, BVDV) is infectious disease caused by virus, respectively
The ox at kind of age all easy infection, with young age ox neurological susceptibility highest.Source of infection is mainly ill domestic animal.This disease is in global distribution, respectively
There is prevalence in cattle-raising developed country, as the disease has become one of Infectious Diseases in American cattle field.China is from import Niu Dangzhong
It has also been found that there is higher seroprevalence, and separating for several times is to virus, it is believed that BVDV, which is ox, has great economy meaning
One of disease, and one of the disease of guard key is answered in import quarantine.
The exploitation of in-vitro diagnosis core material is to develop the premise of corresponding diagnostic reagent, only develop with high activity,
High performance antigen-antibody, external diagnosis reagent product can just become a reality.Therefore, for bovine viral diarrhoea antibody and antigen
Developing can be extremely urgent to produce the raw material of diagnostic reagent, and to some degree, the exploitation of raw material will also become
The primary of bovine viral diarrhea sprawling is reduced to solve the problems, such as.
The content of the invention
It is an object of the invention to provide a kind of bovine viral diarrhoea recombinant protein, its preparation method and applications, are prepared for
With reference to the recombinant protein of the high activity of bovine viral diarrhoea antibody and antigen, high sensitivity and high specific, avoid between raw material
Cross reaction reduce the input of production cost simultaneously.
To achieve the above object, the present invention provides following technical solution：
A kind of bovine viral diarrhoea recombinant protein, which is characterized in that including bovine viral diarrhoea antigen and bovine viral abdomen
Rush down single-chain antibody：
The bovine viral diarrhoea antigen is the E2 antigens containing major antigenic determinant；
The bovine viral diarrhoea single-chain antibody is scFv.
A kind of preparation method of bovine viral diarrhoea recombinant protein, which is characterized in that comprise the following steps：
(1) the E2 antigens containing major antigenic determinant in bovine viral diarrhoea are searched；
(2) codon of bovine viral diarrhoea E2 antigen genes is optimized, synthetic nucleic acid sequence is simultaneously expressed；
(3) structure is directed to the single-chain antibody of bovine viral diarrhoea E2 antigens, transfers the gene of single-chain antibody scFv；
(4) the bovine viral diarrhoea E2 antigen genes of restructuring optimization and the single-chain antibody for bovine viral diarrhoea E2 antigens
The gene of scFv, and expressed；
(5) purifying and renaturation bovine viral diarrhoea recombinant protein.
Further, the step (3) specifically includes herein below：
(31) acquisition of cell strain of monoclonal antibody；
(32) extraction of cell RNA；
(33) using reverse transcription cDNA as template, heavy chain light chain gene is expanded with RT-PCR.
Further, the step (4) specifically includes herein below：
(41) there will be bovine viral diarrhoea E2 antigen genes and for bovine viral diarrhoea E2 antigens by recursive PCR
Single-chain antibody gene links together, and after carrying out double digestion with NdeI and XhoI restriction enzymes, is inserted into identical two
In the pET30a carriers of a digestion processing；
(42) expression vector of above-mentioned recombinant protein gene is converted into e. coli bl21, be coated on containing 100ug/ml
On the LB tablets of kanamycin sulfate, 37 DEG C are incubated overnight, picking monoclonal bacterium colony, and with the sulfuric acid card containing same concentrations, that is mould
37 DEG C of the 300mlLB culture mediums culture of element is to OD600 up to carrying out induced expression after 0.6；After induction, by 4 DEG C of culture solution
5000rpm centrifugations 20min collects thalline.
Further, the step (2) and step (4) are expressed in E. coli system.
Further, the inducing temperature when step (2) and step (4) are expressed is 20 DEG C, 25 DEG C, 28 DEG C or 37 DEG C,
Induction rotating speed is 250rpm, and the IPTG concentration of induction is 0.1mM.
Further, the inducing temperature is 37 DEG C.
Further, the way of purification of the step (5) is selected from ion-exchange chromatography, gel permeation chromatography or affine layer
Analysis.
Further, the way of purification of the step (5) is affinity chromatography.
A kind of application of bovine viral diarrhoea recombinant protein, which is characterized in that the recombinant protein is used to prepare bovine viral
Property diarrhea detection kit.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) exploitation for the first time is for the single-chain antibody scFv of bovine viral diarrhoea major antigen E2.
The present invention carries out recombinant antibodies technological development single-chain antibody again on the basis of monoclonal antibody is developed, single-stranded anti-
Body has the following advantages：1) there is complete antigen binding site, without antibody constant region；2) molecular weight is small, reduces antibody
Non-specific binding, reduce the false positive risks of diagnostic products；3) it is simple in structure, it is easy to genetic engineering transformation；4) it is easy to
Express, can be expressed in eucaryon or prokaryotic cell in cell, can mass production, cost is relatively low.
(2) bovine viral diarrhoea Main Antigenic and the scFv recombinant proteins for E2 antigens are developed for the first time both at home and abroad.
Since infection bovine viral diarrhoea initial stage does not generate antibody, us in this time is caused to can't detect corresponding ox
Virus diarrhea antibody, so as to cause missing inspection, in order to solve the problems, such as that this must increase while bovine viral diarrhoea antibody is detected
Add the detection to antigen, exploitation becomes key for the antibody of bovine viral diarrhoea E2 antigens.
(3) it is prepared in combination with the high activity of bovine viral diarrhoea antibody and antigen, high sensitivity and high specific
Recombinant protein.
After developing in Main Antigenic and for the antibody of bovine viral diarrhoea E2 antigens, technique for gene engineering is utilized
By the efficient epitope filtered out (101-374aa of bovine viral diarrhoea E2 antigens) and for bovine viral diarrhoea E2 antigens
Single-chain antibody scFv (23-30aa) restructuring, be prepared into in combination with the high activity of bovine viral diarrhoea antibody and antigen,
Highly sensitive and high specific recombinant protein, the recombinant protein have the following advantages：1) only need to make in reagent strip is examined soon
It, so can be to avoid the cross reaction between plurality of raw materials with a kind of raw material；2) in the production of raw material, it is only necessary to stablize one
The production technology of kind raw material can so reduce the input of production cost, shorten the raw material production cycle, between reduction production batch
Difference；3) cost of exploitation bovine viral diarrhoea diagnostic reagent is saved.
Description of the drawings
Fig. 1 is the synthesis schematic diagram of the first chains of cDNA.
Specific embodiment
The technical solution in the embodiment of the present invention is clearly and completely described below, it is clear that described embodiment
Only part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel all other embodiments obtained without making creative work belong to the model that the present invention protects
It encloses.
The present embodiment provides a kind of bovine viral diarrhoea recombinant protein, including bovine viral diarrhoea antigen and bovine viral abdomen
Rush down single-chain antibody：
The bovine viral diarrhoea antigen is the E2 antigens containing major antigenic determinant；
The bovine viral diarrhoea single-chain antibody is scFv.
The present embodiment provides a kind of preparation methods of bovine viral diarrhoea recombinant protein, comprise the following steps：
(1) the E2 antigens containing major antigenic determinant in bovine viral diarrhoea are searched；
(2) codon of bovine viral diarrhoea E2 antigen genes is optimized, synthetic nucleic acid sequence is simultaneously expressed；In this implementation
In example, particular content is as follows：
E2 antigen genes GenBank is searched from NCBI：EU159703.1,101-374aa protein sequence are as follows：
FGLCPCDAKPLVRGKFNTTLLNGPAFQMVCPIGWTGTVSCALANKDTLALTVVRTYTRHKPF
PYRQGCITQKTIGEDLYNCDLGGNWTCIPGDQLRYVDGPVESCKWCGYNFYKSEGLPHFPIGKCKL
KNESGYRQVDETSCNRDGVAIVLHGRVKCKIGDTVVQVIAMDDRLGPMPCIPHEIIPSEGPVEKTA
CTFNYTKTLKNKYYEPRDNYFQQYMLKGEYQYWFDLEVTDHHKDYFAESLLVIVVALLGGRYVLWL
LVTYMIISEQTTLG
Sequence is as follows after codon optimization：
TTTGGCCTGTGCCCGTGCGATGCGAAACCGCTGGTGCGCGGCAAATTTAACACCACCCTGCTGAAC
GGCCCGGCGTTTCAGATGGTGTGCCCGATTGGCTGGACCGGCACCGTGAGCTGCGCGCTGGCGAAC
AAAGATACCCTGGCGCTGACCGTGGTGCGCACCTATACCCGCCATAAACCGTTTCCGTATCGCCAG
GGCTGCATTACCCAGAAAACCATTGGCGAAGATCTGTATAACTGCGATCTGGGCGGCAACTGGACC
TGCATTCCGGGCGATCAGCTGCGCTATGTGGATGGCCCGGTGGAAAGCTGCAAATGGTGCGGCTAT
AACTTTTATAAAAGCGAAGGCCTGCCGCATTTTCCGATTGGCAAATGCAAACTGAAAAACGAAAGC
GGCTATCGCCAGGTGGATGAAACCAGCTGCAACCGCGATGGCGTGGCGATTGTGCTGCATGGCCGC
GTGAAATGCAAAATTGGCGATACCGTGGTGCAGGTGATTGCGATGGATGATCGCCTGGGCCCGATG
CCGTGCATTCCGCATGAAATTATTCCGAGCGAAGGCCCGGTGGAAAAAACCGCGTGCACCTTTAAC
TATACCAAAACCCTGAAAAACAAATATTATGAACCGCGCGATAACTATTTTCAGCAGTATATGCTG
AAAGGCGAATATCAGTATTGGTTTGATCTGGAAGTGACCGATCATCATAAAGATTATTTTGCGGAA
AGCCTGCTGGTGATTGTGGTGGCGCTGCTGGGCGGCCGCTATGTGCTGTGGCTGCTGGTGACCTAT
ATGATTATTAGCGAACAGACCACCCTGGGC
It is as follows to design primer：
P1u：CCCATATGTTTGGCCTGTGCCCGTGCGATGCGAAACCGCTGGTGCGCGGCAAATTTAACACC
P1d：CGGGCACACCATCTGAAACGCCGGGCCGTTCAGCAGGGTGGTGTTAAATTTGCCG
P2u：CAGATGGTGTGCCCGATTGGCTGGACCGGCACCGTGAGCTGCGCGCTGGCGAACA
P2d：GGGTATAGGTGCGCACCACGGTCAGCGCCAGGGTATCTTTGTTCGCCAGCGCGC
P3u：TGCGCACCTATACCCGCCATAAACCGTTTCCGTATCGCCAGGGCTGCATTACCCAGA
P3d：GCCCAGATCGCAGTTATACAGATCTTCGCCAATGGTTTTCTGGGTAATGCAGCCC
P4u：AACTGCGATCTGGGCGGCAACTGGACCTGCATTCCGGGCGATCAGCTGCGCTATGT
P4d：TAGCCGCACCATTTGCAGCTTTCCACCGGGCCATCCACATAGCGCAGCTGATC
P5u：CAAATGGTGCGGCTATAACTTTTATAAAAGCGAAGGCCTGCCGCATTTTCCGATTGGC
P5d：CTGGCGATAGCCGCTTTCGTTTTTCAGTTTGCATTTGCCAATCGGAAAATG
P6u：AGCGGCTATCGCCAGGTGGATGAAACCAGCTGCAACCGCGATGGCGTGGCGATTGTG
P6d：CGGTATCGCCAATTTTGCATTTCACGCGGCCATGCAGCACAATCGCCACGCC
P7u：AAATTGGCGATACCGTGGTGCAGGTGATTGCGATGGATGATCGCCTGGGCCCGATGC
P7d：GGCCTTCGCTCGGAATAATTTCATGCGGAATGCACGGCATCGGGCCCAGGCG
P8u：TTCCGAGCGAAGGCCCGGTGGAAAAAACCGCGTGCACCTTTAACTATACCAAAACCCT
P8d：TGAAAATAGTTATCGCGCGGTTCATAATATTTGTTTTTCAGGGTTTTGGTATAG
P9u：CGATAACTATTTTCAGCAGTATATGCTGAAAGGCGAATATCAGTATTGGTTTGATCT
P9d：AGGCTTTCCGCAAAATAATCTTTATGATGATCGGTCACTTCCAGATCAAACCAATAC
P10u：TTTTGCGGAAAGCCTGCTGGTGATTGTGGTGGCGCTGCTGGGCGGCCGCTATGTGCTG
P10d：
GCCCTCGAGGCCCAGGGTGGTCTGTTCGCTAATAATCATATAGGTCACCAGCAGCCACAGCAC
ATAGCGGCC
Experimental method is as follows：
Primer P1u, P1d, P2u, P2d, P3u, P3d are mixed, carry out PCR reactions, products therefrom is named as W1；It will draw
Object P4u, P4d, P5u, P5d, P6u, P6d, P7u, P7d mixing carry out PCR reactions, and products therefrom is named as W2；By primer P8u,
P8d, P9u, P9d, P10u, P10d are mixed, and carry out PCR reactions, and products therefrom is named as W3；Then by above three product W1,
W2, W3 mixing carry out the raq gene of bovine viral diarrhoea after PCR reactions are optimized as template with primer P1u, P10d.
Aforementioned four PCR (TOYOBO Products, article No.：02510D1) reaction condition is as follows：94 DEG C, 2 minutes X, 1 Xun Huan；
(98 DEG C, 15 seconds, 55 DEG C, 30 seconds, 68 DEG C, 30 seconds) X30 Xun Huan；72 DEG C, 7 minutes X, 1 Xun Huan.Sequencing proves that sequence is correct
Afterwards, after this PCR product being carried out double digestion with NdeI and XhoI restriction enzymes (being purchased from NEB companies), it is inserted into and uses phase
In pET30a (Novagen products, article No. 69909-3) carrier with two digestion processing；Step (2) is in E. coli system
Middle expression, inducing temperature during expression are 20 DEG C, 25 DEG C, 28 DEG C or 37 DEG C, and induction rotating speed is 250rpm, the IPTG concentration of induction
For 0.1mM.It is preferred that inducing temperature is 37 DEG C.
(3) structure is directed to the single-chain antibody of bovine viral diarrhoea E2 antigens, transfers the gene of single-chain antibody scFv；Yu Benshi
It applies in example, particular content is as follows：
(31) acquisition of monoclonal antibody (just for the antibody for combining E2 antigen proteins 23-30aa) cell line
A. it will build after successful pET30a-E2 carriers are converted to BL21 expression strain expression and purifications and obtain E2 antigen proteins
B. mouse is immunized with the E2 antigen proteins obtained
C. screened with monoclonal antibody technique just for the antibody cell strain for combining E2 antigen proteins 23-30aa
(32) extraction of cell RNA
With TRIzol LS (invitrogen products, article No. 10296-010) reagent, to specifications operating procedure extraction
Cell total rna.
The synthesis of the first chains of cDNA refer to Fig. 1
(33) using reverse transcription cDNA as template, according to following primer, heavy chain light chain gene is expanded with RT-PCR.
mHVu1:GATGTGAAGCTTCAGGAGTC
mHVu2:CAGGTGCAGCTGAAGGAGTC
mHVu3:CAGGTGCAGCTGAAGCAGTC
mHVu4:CAGGTTACTCTGAAAGAGTC
mHVu5:AAGGTCCAGCTGCAACAATC
mHVu6:GAGGTCCAGCTGCAGCAGTC
mHVu7:CAGGTCCAACTGCAGCAGCC
mHVu8:GAGGTGAAGCTGGTGGAGTC
mHVu9:GAGGTGAAGCTGGTGGAATC
mHVu10:GATGTGAACTTGGAAGTGTC
mHVd1:TGCAGAGACAGTGACCAGAGT
mHVd2:TGAGGAGACTGTGAGAGTGGT
mHVd3:TGAGGAGACGGTGACTGAGGT
mHVd4:TGAGGAGACGGTGACCGTGGT
mKVu1:GATGTTTTGATGACCCAAACT
mKVu2:GATATTGTGATGACGCAGGCT
mKVu3:GATATTGTGATAACCCAG
mKVu4:GACATTGTGCTGACCCAATCT
mKVu5:GACATTGTGATGACCCAGTCT
mKVu6:GATATTGTGCTAACTCAGTCT
mKVu7:GATATCCAGATGACACAGACT
mKVu8:GACATCCAGCTGACTCAGTCT
mKVu9:CAAATTGTTCTCACCCAGTCT
mKVd1:CCGTTTCAGCTCCAGCTTG
mKVd2:CCGTTTTATTTCCAGCTTGGT
mKVd3:CCGTTTTATTTCCAACTTTG
It is combined respectively with mHVd1-mHVd4 with primer mHVu1-mHVu10；MKVu1-mKVu9 respectively with mKVd1-
PCR reactions are done in mKVd4 combinations.
25 μ l of PCR reaction systems, wherein 0.5 μ l of cDNA, 0.25 μ l anti-sense primers of sense primer, 0.25 μ l, Taq enzyme 0.2
μl dNTP 2μl 10×buffer 2.5μl。
PCR (TOYOBO Products, article No.：02510D1), PCR conditions are：94 DEG C, 2 minutes X, 1 Xun Huan；(98
DEG C, 5 seconds, 55 DEG C, 30 seconds, 68 DEG C, 25 seconds) X30 Xun Huan；72 DEG C, 7 minutes X, 1 Xun Huan.
Heavy chain and light chain gene are determined after sequence verification, has then been connected heavy chain with light chain gene by recursive PCR
Come, this recombination is rscFv.
(4) the bovine viral diarrhoea E2 antigen genes (101-374aa) and resist for bovine viral diarrhoea E2 that restructuring optimizes
The gene (23-30aa) of former single-chain antibody scFv, and expressed；In this present embodiment, particular content is as follows：
(41) there will be bovine viral diarrhoea E2 antigen genes (101-374aa) and for bovine viral abdomen by recursive PCR
The single-chain antibody gene (23-30aa) for rushing down E2 antigens links together, (public purchased from NEB with NdeI and XhoI restriction enzymes
Department) carry out double digestion after, be inserted into identical two digestions handle pET30a (Novagen products, article No. 69909-3) load
In body；
(42) expression vector of above-mentioned recombinant protein gene is converted into e. coli bl21, be coated on containing 100ug/ml
Kanamycin sulfate (gives birth to work bioengineering Services Co., Ltd, article No. in Shanghai：KB0286 on LB tablets), 37 DEG C of trainings overnight
It supports, picking monoclonal bacterium colony, is cultivated with 37 DEG C of the 300mlLB culture mediums of the kanamycin sulfate containing same concentrations to OD600
Up to 0.6 or so, the IPTG for being 0.1mM with concentration (gives birth to work, article No.：IB0168 induced expression) is carried out；Inductive condition is as follows, lures
Temperature is led as 20 DEG C, 25 DEG C, 28 DEG C or 37 DEG C, induction rotating speed is 250rpm, induction time 5h, in this present embodiment, preferably
Inducing temperature is 37 DEG C.After induction, 4 DEG C of 5000rpm centrifugations 20min of culture solution are collected into thalline.
(5) purifying and renaturation bovine viral diarrhoea recombinant protein；In this present embodiment, particular content is as follows：
By thalline with 50ml inclusion bodys extract (20mM Tris-HCl, 0.5MUrea pH 7.5,0.5M NaCl, 2%
Triton X-100) it is resuspended, then ultrasonication, condition is each ultrasound 3s, interval 6s, totally 180 times, 12000rpm, 4 DEG C
Inclusion body precipitation is collected by centrifugation.With loading buffer solution B inding Buffer (50mM Tris, 8M Urea, 0.5M Nacl,
20mM imidazoles pH8.0) 50ml crushes inclusion body, upper column purification, way of purification be selected from ion-exchange chromatography, gel permeation chromatography or
Affinity chromatography.In the preferred way of purification of the present embodiment be affinity chromatography；With elution buffer Elution Buffer (50mM
Tris, 8M Urea, 0.5M Nacl, 300mM imidazoles pH8.0) elution destination protein.Recombinant protein after purification is slow with dialysis
Fliud flushing (1mM oxidizeds form of glutathione GSSH, 2mM reduced glutathione GSH, 2mMEDTA, 20mM Tris, pH8.5) is saturating
Analysis, a dialyzate is changed every 48h.The protein liquid after dialysis is taken out, through being concentrated according to ethyl alcohol -20000, in -20 DEG C of preservations
It is spare.
The present embodiment provides a kind of applications of bovine viral diarrhoea recombinant protein, and prepared by recombinant protein bovine viral diarrhoea
Detection kit is used for the detection that bovine viral diarrhoea is immunized.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Profit requirement rather than above description limit, it is intended that all by what is fallen within the meaning and scope of the equivalent requirements of the claims
Variation is included within the present invention.
Claims (10)
1. a kind of bovine viral diarrhoea recombinant protein, which is characterized in that including bovine viral diarrhoea antigen and bovine viral diarrhoea
Single-chain antibody：
The bovine viral diarrhoea antigen is the E2 antigens containing major antigenic determinant；
The bovine viral diarrhoea single-chain antibody is scFv.
2. a kind of preparation method of bovine viral diarrhoea recombinant protein as described in claim 1, which is characterized in that including following
Step：
(1) the E2 antigens containing major antigenic determinant in bovine viral diarrhoea are searched；
(2) codon of bovine viral diarrhoea E2 antigen genes is optimized, synthetic nucleic acid sequence is simultaneously expressed；
(3) structure is directed to the single-chain antibody of bovine viral diarrhoea E2 antigens, transfers the gene of single-chain antibody scFv；
(4) the bovine viral diarrhoea E2 antigen genes of restructuring optimization and the single-chain antibody scFv for bovine viral diarrhoea E2 antigens
Gene, and expressed；
(5) purifying and renaturation bovine viral diarrhoea recombinant protein.
3. the preparation method of bovine viral diarrhoea recombinant protein according to claim 2, which is characterized in that the step
(3) herein below is specifically included：
(31) acquisition of cell strain of monoclonal antibody；
(32) extraction of cell RNA；
(33) using reverse transcription cDNA as template, heavy chain light chain gene is expanded with RT-PCR.
4. the preparation method of bovine viral diarrhoea recombinant protein according to claim 2, which is characterized in that the step
(4) herein below is specifically included：
(41) there will be bovine viral diarrhoea E2 antigen genes and for the single-stranded anti-of bovine viral diarrhoea E2 antigens by recursive PCR
Body gene links together, and after carrying out double digestion with NdeI and XhoI restriction enzymes, is inserted into identical two digestions
In the pET30a carriers of processing；
(42) expression vector of above-mentioned recombinant protein gene is converted into e. coli bl21, is coated on sulfuric acid containing 100ug/ml
On the LB tablets of kanamycins, 37 DEG C are incubated overnight, picking monoclonal bacterium colony, with the kanamycin sulfate containing same concentrations
The culture of 37 DEG C of 300mlLB culture mediums is to OD600 up to carrying out induced expression after 0.6；After induction, by 4 DEG C of 5000rpm of culture solution from
Heart 20min collects thalline.
5. the preparation method of bovine viral diarrhoea recombinant protein according to claim 2, which is characterized in that the step
(2) expressed with step (4) in E. coli system.
6. the preparation method of bovine viral diarrhoea recombinant protein according to claim 2, which is characterized in that the step
(2) inducing temperature when and step (4) is expressed is 20 DEG C, 25 DEG C, 28 DEG C or 37 DEG C, and induction rotating speed is 250rpm, induction
IPTG concentration is 0.1mM.
7. the preparation method of bovine viral diarrhoea recombinant protein according to claim 6, which is characterized in that the induction temperature
It spends for 37 DEG C.
8. the preparation method of bovine viral diarrhoea recombinant protein according to claim 2, which is characterized in that the step
(5) way of purification is selected from ion-exchange chromatography, gel permeation chromatography or affinity chromatography.
9. the preparation method of bovine viral diarrhoea recombinant protein according to claim 8, which is characterized in that the step
(5) way of purification is affinity chromatography.
A kind of 10. application of bovine viral diarrhoea recombinant protein as described in claim 1, which is characterized in that the restructuring egg
It is used to prepare bovine viral diarrhoea detection kit in vain.
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