CN110790827A

CN110790827A - I-type bovine herpes virus recombinant protein and preparation method and application thereof

Info

Publication number: CN110790827A
Application number: CN201911284601.9A
Authority: CN
Inventors: 李晓光; 刘毅
Original assignee: Hangzhou Billion Mino Biological Technology Co Ltd
Current assignee: Hangzhou Billion Mino Biological Technology Co Ltd
Priority date: 2018-12-26
Filing date: 2019-12-13
Publication date: 2020-02-14
Also published as: CN109593121A

Abstract

The invention discloses a recombinant protein of bovine herpes virus type I, which relates to the technical field of biomedicine, and a preparation method of the recombinant protein of bovine herpes virus type I comprises the following steps of (1) searching gD antigen containing main antigenic determinant in the bovine herpes virus type I; (2) optimizing codons of the bovine herpes virus I gD antigen gene, synthesizing a nucleic acid sequence and expressing the nucleic acid sequence; (3) constructing a single-chain antibody aiming at the type I bovine herpes virus gD antigen, and calling the gene of the single-chain antibody scFv; (4) recombining and expressing an optimized gene of the gD antigen of the bovine herpes virus type I and a gene of a single-chain antibody scFv aiming at the gD antigen of the bovine herpes virus type I in an induction manner; (5) purifying and renaturing the bovine herpes virus type I recombinant protein. The invention develops the single-chain antibody by recombinant antibody technology on the basis of developing the monoclonal antibody, is easy to express in cells, can express in eukaryotic cells or prokaryotic cells, can be produced in large quantities, and has lower cost.

Description

I-type bovine herpes virus recombinant protein and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a bovine herpes virus type I recombinant protein.

Background

Infectious Bovine Rhinotracheitis (IBR), also known as necrotic rhinitis and red nose disease, is a bovine respiratory infectious disease caused by bovine herpes virus type I (BHV-1). The disease is prevalent in the world at present, and has great influence on milk yield of dairy cows and reproductive capacity of bulls. The disease is reported from imported dairy cows of New Zealand for the first time in 1980, a strain of infectious bovine rhinotracheitis virus is separated, and then serological investigation proves that IBR viruses exist in black white cow, local yellow cow, buffalo or yak in provinces such as Guangdong, Guangxi and the like in China. In some areas where traffic is extremely inconvenient, the IBR virus antibody positivity rate is extremely high.

The development of the core raw materials for in vitro diagnosis is the premise of developing corresponding diagnostic reagents, and only when antigen antibodies with high activity and high performance are developed, the in vitro diagnostic reagent products can become practical. Therefore, the development of materials for producing diagnostic reagents against bovine herpesvirus type I antibodies and antigens is imminent, and to some extent, the development of materials will be the primary solution to reduce the spread of bovine herpesvirus type I.

Disclosure of Invention

The invention provides a recombinant protein of bovine herpes virus type I, which comprises a bovine herpes virus type I antigen and a bovine herpes virus type I single-chain antibody.

Furthermore, the bovine herpes virus type I antigen is a gD antigen containing a main antigenic determinant, and the bovine herpes virus type I single-chain antibody is scFv.

The invention also provides a preparation method of the I-type bovine herpes virus recombinant protein, which comprises the following preparation steps:

(1) finding gD antigens with major epitopes in bovine herpes virus type I

The gD antigen of bovine herpes virus type I, which contains a major antigenic determinant, was found from NCBI (national center for Biotechnology information).

(2) Optimization of codons of bovine herpes virus type I gD antigen gene, synthesis of nucleic acid sequence and expression thereof

In the expression of proteins in E.coli, the frequencies of different codon usage are greatly different, and in order to maximize the expression amount of recombinant proteins, the type I bovine herpes virus gD antigen gene sequence is subjected to codon optimization, and then an optimized nucleic acid sequence is synthesized and expressed.

(3) Construction of Single-chain antibody against bovine herpes Virus type I gD antigen, and Gene calling of Single-chain antibody scFv

Immunizing a mouse with the purified gD antigen protein to obtain a high-activity monoclonal antibody, and then regulating the gene of the single-chain antibody scFv.

(4) Recombination optimized G D antigen gene of bovine herpes virus type I and single-chain antibody scFv gene aiming at G D antigen of bovine herpes virus type I, and expression

The optimized bovine herpes virus type I gD antigen gene (38-292aa) and a single-chain antibody scFv gene (capable of being directed to a part 23-30aa of a gD antigen binding protein) for the bovine herpes virus type I gD antigen are connected by a recursive PCR technology, and after sequencing analysis, a gene fragment of a recombinant protein is inserted into an expression vector for expression.

(5) Purifying and renaturing the bovine herpes virus type I recombinant protein.

The expression system of the invention is an escherichia coli expression system, and has the characteristics of short period, low cost, large expression quantity and the like.

In order to better maintain the active site of the recombinant protein, the invention selects milder culture and induction conditions, the induction temperature is 20 ℃,25 ℃, 28 ℃ and 37 ℃ when the recombinant protein is expressed, the induction temperature is more preferably 25 ℃ and 37 ℃, the induction temperature is more preferably 25 ℃, the induction speed is 250rpm, and the concentration of induced IPTG (isopropyl- β -D-thiogalactoside) is 0.5 mM., so that the recombinant protein has slower expression and has sufficient time for space conformation formation.

The invention can break the bacteria by adopting an ultrasonic mode. In order to avoid the vigorous conditions, the cells were disrupted at 200w, 6s per sonication and 180 cycles with 3s intervals.

The recombinant protein of the present invention may be purified by ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like, and preferably by affinity chromatography.

According to the invention, the 6XHis tag is added into the recombinant protein, so that the affinity chromatography purification mode can achieve higher purity.

The recombinant protein of the invention utilizes the genetic engineering recombination technology to express the recombinant protein method of the bovine herpes virus type I antigen and the single-chain antibody in an escherichia coli system, and has the advantages of short production period, high yield, low cost and the like.

The recombinant bovine herpes virus type I protein of the invention can be used as a part of a bovine herpes virus type I immunodiagnosis kit.

The invention has the following advantages and beneficial effects:

(1) single chain antibody scFv was first developed against the major antigen gD of bovine herpes virus type I.

The invention develops the single-chain antibody by recombinant antibody technology on the basis of developing the monoclonal antibody, and the single-chain antibody has the following advantages: 1) has complete antigen binding sites and does not contain antibody constant regions; 2) the molecular weight is small, the non-specific binding of the antibody is reduced, and the false positive risk of a diagnostic product is reduced; 3) the structure is simple, and the genetic engineering transformation is easy; 4) easy to express in cells, can express in eukaryotic or prokaryotic cells, can be produced in large quantities and has lower cost.

(2) The main epitope of the bovine herpes virus type I and the scFv recombinant protein aiming at the gD antigen are firstly developed at home and abroad.

Since no antibody is produced in the early stage of infection with bovine herpes virus type I, no corresponding bovine herpes virus type I antibody can be detected in the period of time, thus resulting in omission, and in order to solve the problem, detection of bovine herpes virus type I antibody and detection of antigen must be increased, and development of antibody against bovine herpes virus type I gD antigen becomes key.

(3) Recombinant proteins are produced that simultaneously bind bovine herpes virus type I antibodies and antigens with high activity, high sensitivity and high specificity.

After the main antigen epitope and the antibody aiming at the bovine herpes virus type I gD antigen are developed, the screened high-efficiency antigen epitope (38-292aa of the bovine herpes virus type I gD antigen) and the single-chain antibody scFv (23-30aa) aiming at the bovine herpes virus type I gD antigen are recombined by utilizing a genetic engineering technology to prepare a recombinant protein which has high activity, high sensitivity and high specificity and simultaneously combines the bovine herpes virus type I antibody and the antigen, and the recombinant protein has the following advantages: 1) only one raw material is needed to be used in the quick diagnosis reagent strip, so that the cross reaction among various raw materials can be avoided; 2) in the production of raw materials, only one production process of the raw materials needs to be stabilized, so that the investment of production cost can be reduced, the production period of the raw materials is shortened, and the production batch difference is reduced; 3) saves the cost of developing the diagnostic reagent for bovine herpes virus type I.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the invention thereto. It will be appreciated by those skilled in the art that the present invention encompasses all alternatives, modifications and equivalents as may be included within the scope of the claims.

Example 1 codon optimization of bovine herpes virus type I gD antigen Gene and expression vector construction

Search gD antigen gene GenBank from NCBI: AFB76672.1, the 38-292aa protein sequence of which is as follows: PRYNYTERWHTTGPIPSPFADGREQPVEVRYATSAAACDMLALIADPQVGRTLWEAVRRHARAYNATVIWYKIESGCARPLY YMEYTECEPRKHFGYCRYRTPPFWDSFLAGFAYPTDDELGLIMAAPARLVEGQYRRALYIDGTVAYTDFMVSLPAGDCWFS KLGAARGYTFGACFPARDYEQKKVLRLTYLTQYYPQEAHKAIVDYWFMRHGGVVPPYFEESKGYEPPPAADGGSPAPPGD DEAREDEGETED

The sequence after codon optimization is as follows:

CCTCGTTATAATTACACCGAACGCTGGCATACCACCGGTCCGATTCCGAGCCCGTTTGCCGATGGCCGCGAA CAGCCGGTGGAAGTTCGTTATGCAACCAGTGCAGCCGCCTGCGATATGCTGGCCCTGATTGCCGATCCGCAGGTGGGTCGCACCCTGTGGGAAGCAGTGCGTCGTCATGCACGCGCCTATAATGCAACCGTTATTTGGTATAA AATCGAAAGTGGCTGTGCACGCCCGCTGTATTATATGGAATATACCGAATGTGAACCGCGCAAACATTTTGG TTATTGCCGTTATCGTACCCCGCCGTTTTGGGATAGCTTTCTGGCAGGCTTTGCCTATCCGACCGATGATGAA CTGGGTCTGATTATGGCCGCACCGGCCCGTCTGGTGGAAGGCCAATATCGTCGCGCACTGTATATTGATGGC ACCGTTGCATATACCGATTTTATGGTTAGTCTGCCGGCCGGCGATTGCTGGTTTAGTAAACTGGGTGCCGCA CGTGGTTATACCTTTGGCGCATGCTTTCCGGCCCGTGATTATGAACAGAAAAAAGTGCTGCGCCTGACCTAT CTGACCCAGTATTATCCGCAGGAAGCCCATAAAGCAATTGTGGATTATTGGTTTATGCGCCATGGCGGCGTT GTTCCGCCGTATTTTGAAGAAAGCAAAGGCTATGAACCGCCGCCGGCAGCAGATGGTGGTAGTCCGGCAC CGCCGGGTGACGATGAAGCCCGTGAAGATGAAGGTGAAACCGAAGAT

primers were designed as follows:

P1u：GCCCATATGCCTCGTTATAATTACACCGAACGCTGGCATACCACCGGTCCGATTCCG

P1d：ATAACGAACTTCCACCGGCTGTTCGCGGCCATCGGCAAACGGGCTCGGAATCGGACCGGT

P2u：GTGGAAGTTCGTTATGCAACCAGTGCAGCCGCCTGCGATATGCTGGCCCTGATTGCCG

P2d：GACGACGCACTGCTTCCCACAGGGTGCGACCCACCTGCGGATCGGCAATCAGGGCCA

P3u：AAGCAGTGCGTCGTCATGCACGCGCCTATAATGCAACCGTTATTTGGTATAAAATC

P3d：GGTATATTCCATATAATACAGCGGGCGTGCACAGCCACTTTCGATTTTATACCAAAT

P4u：TATATGGAATATACCGAATGTGAACCGCGCAAACATTTTGGTTATTGCCGTTATCGTAC

P4d：CGGATAGGCAAAGCCTGCCAGAAAGCTATCCCAAAACGGCGGGGTACGATAACGGCAA

P5u：AGGCTTTGCCTATCCGACCGATGATGAACTGGGTCTGATTATGGCCGCACCGGCC

P5d：GCCATCAATATACAGTGCGCGACGATATTGGCCTTCCACCAGACGGGCCGGTGCGGCCAT

P6u：ACTGTATATTGATGGCACCGTTGCATATACCGATTTTATGGTTAGTCTGCCGGCCGGCG

P6d：AAAGGTATAACCACGTGCGGCACCCAGTTTACTAAACCAGCAATCGCCGGCCGGCAGAC

P7u：TTTGGCGCATGCTTTCCGGCCCGTGATTATGAACAGAAAAAAGTGCTGCGCCTGACCTAT

P7d：CCACAATTGCTTTATGGGCTTCCTGCGGATAATACTGGGTCAGATAGGTCAGGCGCAG

P8u：ATAAAGCAATTGTGGATTATTGGTTTATGCGCCATGGCGGCGTTGTTCCGCCGTATT

P8d：CCATCTGCTGCCGGCGGCGGTTCATAGCCTTTGCTTTCTTCAAAATACGGCGGAACAA

P9u：GCCGGCAGCAGATGGTGGTAGTCCGGCACCGCCGGGTGACGATGAAGCCCGT

P9d：GCCCTCGAGATCTTCGGTTTCACCTTCATCTTCACGGGCTTCATCGTC

the experimental method is as follows:

mixing the primers P1u, P1d, P2u, P2d, P3u and P3d, carrying out PCR reaction, and naming the obtained product as W1; mixing the primers P4u, P4d, P5u, P5d, P6u and P6d, carrying out PCR reaction, and naming the obtained product as W2; mixing the primers P7u, P7d, P8u, P8d, P9u and P9d, carrying out PCR reaction, and naming the obtained product as W3; then, the three products W1, W2 and W3 were mixed as a template, and PCR was carried out using primers P1u and P9d to obtain the gD gene of the optimized bovine herpes virus type I. The four PCR (TOYOBO Co., Ltd., cat # 02510D1) reaction conditions were as follows: 94 ℃,2 min X1 cycles; (98 ℃,15 seconds, 55 ℃,30 seconds, 68 ℃,30 seconds) X30 cycles; 72 ℃,7 min X1 cycles. After the sequence was confirmed by sequencing, the PCR product was double-digested with NdeI and XhoI restriction enzymes (available from NEB), and inserted into pET30a (Novagen, cat. No. 69909-3) vector treated with the same two digestions.

Example 2 construction of Single chain antibodies against bovine herpes Virus type I core protein bovine herpes Virus gD antigen

(1) Acquisition of monoclonal antibody (directed against antibody binding to gD antigen protein 23-30aa only) cell line

a. Transforming the successfully constructed pET30a-gD vector into BL21 expression strain for expression and purification to obtain gD antigen protein

b. Immunization of mice with the gD antigen protein obtained

c. Monoclonal antibody technology is used for screening antibody cell strains only aiming at combined gD antigen protein 23-30aa

(2) Extraction of cellular RNA

Total cellular RNA was extracted using TRIzol LS (Invitrogen products, cat. No. 10296-010) reagent according to the protocol.

First Strand cDNA Synthesis

Taking 1 mu gRNA as a reverse transcription template, oligo (d) T1 mu l as a reverse transcription primer,

adding DEPC water to the total volume of 12 mu l, and uniformly mixing;

denaturation at 70 deg.C for 5min, and rapidly cooling in ice bath;

adding 5 × buffer solution 4 μ l, RNase inhibitor 1 μ l, dNTP (10mMeach)2 μ l, and mixing

Incubating at 37 ℃ for 5 min;

adding 1 μ l of AMV reverse transcriptase;

reverse transcription is carried out for 60min at 37 ℃;

terminating the reaction at 70 ℃ for 10 min;

and (5) cooling on ice.

(3) The heavy chain and light chain genes were amplified by RT-PCR using the reverse transcribed cDNA as a template and the following primers.

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

Primers mHVu 1-mHVu 10 were used in combination with mHVd 1-mHVd 4, respectively; mKVu 1-mKVu 9 were combined with mKVd 1-mKVd 4, respectively, for PCR.

The PCR reaction system contained 25. mu.l of cDNA0.5. mu.l, upstream primer 0.25. mu.l of downstream primer 0.25. mu.l, Taq enzyme 0.2. mu.l of NTP 2. mu.l 10 Xbuffer 2.5. mu.l.

PCR (TOYOBO Co., Ltd., cat # 02510D1) under the conditions: 94 ℃,2 min X1 cycles; (98 ℃,5 seconds, 55 ℃,30 seconds, 68 ℃,25 seconds) X30 cycles; 72 ℃,7 min X1 cycles.

After sequencing verification, the heavy chain and light chain genes are determined, and then the heavy chain and light chain genes are connected through recursive PCR, and the recombinant gene is the rscFv. The sequence of the rscFv was as follows:

GAGGTGAAGCTGGTGGAATCTGGCGCTGAGGTGAAGAAGCCTGGCTCCTCGGTGAAGGTCT CCTGCACCAGCAGCGAAGTGACCTTCAGCAGCTTCGCTATCAGCTGGGTGCGCCAGGCCCC GGGTCAAGGCCTGGAGTGGCTGGGTGGTATTAGCCCGATGTTTGGCACCCCGAACTATGCAC AGAAGTTCCAGGGCCGCGTCACGATTACCGCGGACCAGAGCACCCGCACCGCGTATATGGA TCTGCGCAGCCTGCGCTCTGAGGACACGGCCGTGTATTACTGTGCGCGCAGCCCGAGCTATA TTTGCAGCGGCGGCACCTGCGTGTTTGATCATTGGGGCCAGGGCACCTCAGTCACCGTCTCC TCAGGCGGCGGCGGCAGCGATATTGTGATAACCCAGCCGTCGGTGTCCAAGGGCTTGCGCC AGACCGCCACACTGACCTGCACTGGTAACAGCAACAATGTGGGCAACCAAGGTGCAGCTTG GCTGCAGCAGCACCAGGGCCACCCTCCCAAACTGCTGTCCTACAGGAATAACGATCGCCCC TCAGGTATTTCAGAGCGCTTTTCTGCATCCCGCTCAGGTAACACAGCCTCCCTGACCATTACT GGCCTGCAGCCTGAGGACGAGGCTGACTATTACTGCTCAACCTGGGACAGCAGCCTCAGTG CTGTGGTGTTCGGCGGCACAAAGTTGGAAATAAAACGG

example 3 recombination of a Gene containing bovine herpes virus type I gD antigen (38-292aa) and a Gene of a Single chain antibody against bovine herpes virus type I gD antigen (23-30aa), expression of the gene

(1) A gene containing bovine herpes virus type I gD antigen (38-292aa) and a gene of a single-chain antibody against bovine herpes virus type I gD antigen (23-30aa) were ligated together by recursive PCR, and after double cleavage with NdeI and XhoI restriction enzymes (available from NEB), they were inserted into pET30a (Novagen product, cat # 69909-3) vector treated with the same two cleavage enzymes. The gD-rscFv fusion sequence is as follows:

CCTCGTTATAATTACACCGAACGCTGGCATACCACCGGTCCGATTCCGAGCCCGTTTGCCGAT GGCCGCGAACAGCCGGTGGAAGTTCGTTATGCAACCAGTGCAGCCGCCTGCGATATGCTGG CCCTGATTGCCGATCCGCAGGTGGGTCGCACCCTGTGGGAAGCAGTGCGTCGTCATGCACG CGCCTATAATGCAACCGTTATTTGGTATAAAATCGAAAGTGGCTGTGCACGCCCGCTGTATTA TATGGAATATACCGAATGTGAACCGCGCAAACATTTTGGTTATTGCCGTTATCGTACCCCGCC GTTTTGGGATAGCTTTCTGGCAGGCTTTGCCTATCCGACCGATGATGAACTGGGTCTGATTAT GGCCGCACCGGCCCGTCTGGTGGAAGGCCAATATCGTCGCGCACTGTATATTGATGGCACCG TTGCATATACCGATTTTATGGTTAGTCTGCCGGCCGGCGATTGCTGGTTTAGTAAACTGGGTG CCGCACGTGGTTATACCTTTGGCGCATGCTTTCCGGCCCGTGATTATGAACAGAAAAAAGTG CTGCGCCTGACCTATCTGACCCAGTATTATCCGCAGGAAGCCCATAAAGCAATTGTGGATTAT TGGTTTATGCGCCATGGCGGCGTTGTTCCGCCGTATTTTGAAGAAAGCAAAGGCTATGAACC GCCGCCGGCAGCAGATGGTGGTAGTCCGGCACCGCCGGGTGACGATGAAGCCCGTGAAGAT GAAGGTGAAACCGAAGATGAGGTGAAGCTGGTGGAATCTGGCGCTGAGGTGAAGAAGCCT GGCTCCTCGGTGAAGGTCTCCTGCACCAGCAGCGAAGTGACCTTCAGCAGCTTCGCTATCA GCTGGGTGCGCCAGGCCCCGGGTCAAGGCCTGGAGTGGCTGGGTGGTATTAGCCCGATGTT TGGCACCCCGAACTATGCACAGAAGTTCCAGGGCCGCGTCACGATTACCGCGGACCAGAGC ACCCGCACCGCGTATATGGATCTGCGCAGCCTGCGCTCTGAGGACACGGCCGTGTATTACTG TGCGCGCAGCCCGAGCTATATTTGCAGCGGCGGCACCTGCGTGTTTGATCATTGGGGCCAGGGCACCTCAGTCACCGTCTCCTCAGGCGGCGGCGGCAGCGATATTGTGATAACCCAGCCGTCG GTGTCCAAGGGCTTGCGCCAGACCGCCACACTGACCTGCACTGGTAACAGCAACAATGTGG GCAACCAAGGTGCAGCTTGGCTGCAGCAGCACCAGGGCCACCCTCCCAAACTGCTGTCCTA CAGGAATAACGATCGCCCCTCAGGTATTTCAGAGCGCTTTTCTGCATCCCGCTCAGGTAACA CAGCCTCCCTGACCATTACTGGCCTGCAGCCTGAGGACGAGGCTGACTATTACTGCTCAACC TGGGACAGCAGCCTCAGTGCTGTGGTGTTCGGCGGCACAAAGTTGGAAATAAAACGG

(2) the expression vector of the recombinant protein gene was transformed into Escherichia coli BL21, spread on an LB plate containing 100ug/ml kanamycin sulfate (Shanghai Biotech services Co., Ltd.; product No.: KB0286), cultured overnight at 37 ℃, and a single colony was picked up, cultured at 37 ℃ in 300ml LB medium containing the same concentration of kanamycin sulfate to an OD600 of about 0.6, and induced to express using IPTG (Bio-worker, product No.: IB0168) at a concentration of 0.5mM under the induction conditions: 37 ℃ and 250rpm for 5 h. After induction, the culture was centrifuged at 5000rpm at 4 ℃ for 20min to collect the cells.

Example 4 purification and renaturation of recombinant proteins containing bovine herpes virus type I

The cells were resuspended in 50ml of inclusion body extract (20mM Tris-HCl,0.5MureapH7.5,0.5M NaCl, 2% Triton X-100) and then sonicated under conditions of 3s per sonication, 6s intervals, 180 total times, 12000rpm, and centrifugation at 4 ℃ to collect inclusion body pellets. The inclusion bodies were disrupted in 50ml of loading buffer (50mM Tris,8MUrea,0.5M Nacl,20mM imidazole pH8.0), purified by column chromatography, and the objective protein was eluted in elution buffer ElutionBuffer (50mM Tris,8MUrea,0.5M Nacl,300mM imidazole pH 8.0). The purified recombinant protein was dialyzed against a dialysis buffer (1mM oxidized glutathione GSSH,2mM reduced glutathione GSH,2mM EDTA,20mM Tris, pH8.5) and the dialysate was changed every 48 hours. Taking out dialyzed protein solution, concentrating with ethanol-20000, and storing at-20 deg.C.

SEQUENCE LISTING

<110> Hangzhou Yiminou Biotechnology Ltd

<120> type I bovine herpes virus recombinant protein, preparation method and application thereof

<130>2

<160>46

<170>PatentIn version 3.3

<210>1

<211>255

<212>PRT

<213> GenBank of gD antigen gene: 38-292aa protein sequence of (AFB 76672.1)

<400>1

Pro Arg Tyr Asn Tyr Thr Glu Arg Trp His Thr Thr Gly Pro Ile Pro

1 5 10 15

Ser Pro Phe Ala Asp Gly Arg Glu Gln Pro Val Glu Val Arg Tyr Ala

20 25 30

Thr Ser Ala Ala Ala Cys Asp Met Leu Ala Leu Ile Ala Asp Pro Gln

35 40 45

Val Gly Arg Thr Leu Trp Glu Ala Val Arg Arg His Ala Arg Ala Tyr

50 55 60

Asn Ala Thr Val Ile Trp Tyr Lys Ile Glu Ser Gly Cys Ala Arg Pro

65 70 75 80

Leu Tyr Tyr Met Glu Tyr Thr Glu Cys Glu Pro Arg Lys His Phe Gly

85 90 95

Tyr Cys Arg Tyr Arg Thr Pro Pro Phe Trp Asp Ser Phe Leu Ala Gly

100 105 110

Phe Ala Tyr Pro Thr Asp Asp Glu Leu Gly Leu Ile Met Ala Ala Pro

115 120 125

Ala Arg Leu Val Glu Gly Gln Tyr Arg Arg Ala Leu Tyr Ile Asp Gly

130 135 140

Thr Val Ala Tyr Thr Asp Phe Met Val Ser Leu Pro Ala Gly Asp Cys

145 150 155 160

Trp Phe Ser Lys Leu Gly Ala Ala Arg Gly Tyr Thr Phe Gly Ala Cys

165 170 175

Phe Pro Ala Arg Asp Tyr Glu Gln Lys Lys Val Leu Arg Leu Thr Tyr

180 185 190

Leu Thr Gln Tyr Tyr Pro Gln Glu Ala His Lys Ala Ile Val Asp Tyr

195 200 205

Trp Phe Met Arg His Gly Gly Val Val Pro Pro Tyr Phe Glu Glu Ser

210 215 220

Lys Gly Tyr Glu Pro Pro Pro Ala Ala Asp Gly Gly Ser Pro Ala Pro

225 230 235 240

Pro Gly Asp Asp Glu Ala Arg Glu Asp Glu Gly Glu Thr Glu Asp

245 250 255

<210>2

<211>765

<212>PRT

<213> GenBank of gD antigen gene: (AFB 76672.1) 38-292aa protein sequence after codon optimization

<400>2

Cys Cys Thr Cys Gly Thr Thr Ala Thr Ala Ala Thr Thr Ala Cys Ala

1 5 10 15

Cys Cys Gly Ala Ala Cys Gly Cys Thr Gly Gly Cys Ala Thr Ala Cys

20 25 30

Cys Ala Cys Cys Gly Gly Thr Cys Cys Gly Ala Thr Thr Cys Cys Gly

35 40 45

Ala Gly Cys Cys Cys Gly Thr Thr Thr Gly Cys Cys Gly Ala Thr Gly

50 55 60

Gly Cys Cys Gly Cys Gly Ala Ala Cys Ala Gly Cys Cys Gly Gly Thr

65 70 7580

Gly Gly Ala Ala Gly Thr Thr Cys Gly Thr Thr Ala Thr Gly Cys Ala

85 90 95

Ala Cys Cys Ala Gly Thr Gly Cys Ala Gly Cys Cys Gly Cys Cys Thr

100 105 110

Gly Cys Gly Ala Thr Ala Thr Gly Cys Thr Gly Gly Cys Cys Cys Thr

115 120 125

Gly Ala Thr Thr Gly Cys Cys Gly Ala Thr Cys Cys Gly Cys Ala Gly

130 135 140

Gly Thr Gly Gly Gly Thr Cys Gly Cys Ala Cys Cys Cys Thr Gly Thr

145 150 155 160

Gly Gly Gly Ala Ala Gly Cys Ala Gly Thr Gly Cys Gly Thr Cys Gly

165 170 175

Thr Cys Ala Thr Gly Cys Ala Cys Gly Cys Gly Cys Cys Thr Ala Thr

180 185 190

Ala Ala Thr Gly Cys Ala Ala Cys Cys Gly Thr Thr Ala Thr Thr Thr

195 200 205

Gly Gly Thr Ala Thr Ala Ala Ala Ala Thr Cys Gly Ala Ala Ala Gly

210 215 220

Thr Gly Gly Cys Thr Gly Thr Gly Cys Ala Cys Gly Cys Cys Cys Gly

225 230 235 240

Cys Thr Gly Thr Ala Thr Thr Ala Thr Ala Thr Gly Gly Ala Ala Thr

245 250 255

Ala Thr Ala Cys Cys Gly Ala Ala Thr Gly Thr Gly Ala Ala Cys Cys

260 265 270

Gly Cys Gly Cys Ala Ala Ala Cys Ala Thr Thr Thr Thr Gly Gly Thr

275 280 285

Thr Ala Thr Thr Gly Cys Cys Gly Thr Thr Ala Thr Cys Gly Thr Ala

290 295 300

Cys Cys Cys Cys Gly Cys Cys Gly Thr Thr Thr Thr Gly Gly Gly Ala

305 310 315 320

Thr Ala Gly Cys Thr Thr Thr Cys Thr Gly Gly Cys Ala Gly Gly Cys

325 330 335

Thr Thr Thr Gly Cys Cys Thr Ala Thr Cys Cys Gly Ala Cys Cys Gly

340 345 350

Ala Thr Gly Ala Thr Gly Ala Ala Cys Thr Gly Gly Gly Thr Cys Thr

355 360 365

Gly Ala Thr Thr Ala Thr Gly Gly Cys Cys Gly Cys Ala Cys Cys Gly

370 375 380

Gly Cys Cys Cys Gly Thr Cys Thr Gly Gly Thr Gly Gly Ala Ala Gly

385 390 395 400

Gly Cys Cys Ala Ala Thr Ala Thr Cys Gly Thr Cys Gly Cys Gly Cys

405 410 415

Ala Cys Thr Gly Thr Ala Thr Ala Thr Thr Gly Ala Thr Gly Gly Cys

420 425 430

Ala Cys Cys Gly Thr Thr Gly Cys Ala Thr Ala Thr Ala Cys Cys Gly

435 440 445

Ala Thr Thr Thr Thr Ala Thr Gly Gly Thr Thr Ala Gly Thr Cys Thr

450 455 460

Gly Cys Cys Gly Gly Cys Cys Gly Gly Cys Gly Ala Thr Thr Gly Cys

465 470 475 480

Thr Gly Gly Thr Thr Thr Ala Gly Thr Ala Ala Ala Cys Thr Gly Gly

485 490 495

Gly Thr Gly Cys Cys Gly Cys Ala Cys Gly Thr Gly Gly Thr Thr Ala

500 505 510

Thr Ala Cys Cys Thr Thr Thr Gly Gly Cys Gly Cys Ala Thr Gly Cys

515 520 525

Thr Thr Thr Cys Cys Gly Gly Cys Cys Cys Gly Thr Gly Ala Thr Thr

530 535 540

Ala Thr Gly Ala Ala Cys Ala Gly Ala Ala Ala Ala Ala Ala Gly Thr

545 550 555 560

Gly Cys Thr Gly Cys Gly Cys Cys Thr Gly Ala Cys Cys Thr Ala Thr

565 570 575

Cys Thr Gly Ala Cys Cys Cys Ala Gly Thr Ala Thr Thr Ala Thr Cys

580 585 590

Cys Gly Cys Ala Gly Gly Ala Ala Gly Cys Cys Cys Ala Thr Ala Ala

595 600 605

Ala Gly Cys Ala Ala Thr Thr Gly Thr Gly Gly Ala Thr Thr Ala Thr

610 615 620

Thr Gly Gly Thr Thr Thr Ala Thr Gly Cys Gly Cys Cys Ala Thr Gly

625 630 635 640

Gly Cys Gly Gly Cys Gly Thr Thr Gly Thr Thr Cys Cys Gly Cys Cys

645 650 655

Gly Thr Ala Thr Thr Thr Thr Gly Ala Ala Gly Ala Ala Ala Gly Cys

660 665 670

Ala Ala Ala Gly Gly Cys Thr Ala Thr Gly Ala Ala Cys Cys Gly Cys

675 680 685

Cys Gly Cys Cys Gly Gly Cys Ala Gly Cys Ala Gly Ala Thr Gly Gly

690 695 700

Thr Gly Gly Thr Ala Gly Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly

705 710 715 720

Cys Cys Gly Gly Gly Thr Gly Ala Cys Gly Ala Thr Gly Ala Ala Gly

725 730 735

Cys Cys Cys Gly Thr Gly Ala Ala Gly Ala Thr Gly Ala Ala Gly Gly

740 745 750

Thr Gly Ala Ala Ala Cys Cys Gly Ala Ala Gly Ala Thr

755 760 765

<210>3

<211>57

<212>DNA

<213> design of primer (P1 u)

<400>3

gcccatatgc ctcgttataa ttacaccgaa cgctggcata ccaccggtcc gattccg 57

<210>4

<211>60

<212>DNA

<213> design of primer (P1 d)

<400>4

ataacgaact tccaccggct gttcgcggcc atcggcaaac gggctcggaa tcggaccggt 60

<210>5

<211>58

<212>DNA

<213> design of primer (P2 u)

<400>5

gtggaagttc gttatgcaac cagtgcagcc gcctgcgata tgctggccct gattgccg 58

<210>6

<211>57

<212>DNA

<213> design of primer (P2 d)

<400>6

gacgacgcac tgcttcccac agggtgcgac ccacctgcgg atcggcaatc agggcca 57

<210>7

<211>56

<212>DNA

<213> design of primer (P3 u)

<400>7

aagcagtgcg tcgtcatgca cgcgcctata atgcaaccgt tatttggtat aaaatc 56

<210>8

<211>57

<212>DNA

<213> design of primer (P3 d)

<400>8

ggtatattcc atataataca gcgggcgtgc acagccactt tcgattttat accaaat 57

<210>9

<211>59

<212>DNA

<213> design of primer (P4 u)

<400>9

tatatggaat ataccgaatg tgaaccgcgc aaacattttg gttattgccg ttatcgtac 59

<210>10

<211>58

<212>DNA

<213> design of primer (P4 d)

<400>10

cggataggca aagcctgcca gaaagctatc ccaaaacggc ggggtacgat aacggcaa 58

<210>11

<211>55

<212>DNA

<213> design of primer (P5 u)

<400>11

aggctttgcc tatccgaccg atgatgaact gggtctgatt atggccgcac cggcc 55

<210>12

<211>60

<212>DNA

<213> design of primer (P5 d)

<400>12

gccatcaata tacagtgcgc gacgatattg gccttccacc agacgggccg gtgcggccat 60

<210>13

<211>59

<212>DNA

<213> design of primer (P6 u)

<400>13

actgtatatt gatggcaccg ttgcatatac cgattttatg gttagtctgc cggccggcg 59

<210>14

<211>59

<212>DNA

<213> design of primer (P6 d)

<400>14

aaaggtataa ccacgtgcgg cacccagttt actaaaccag caatcgccgg ccggcagac 59

<210>15

<211>60

<212>DNA

<213> design of primer (P7 u)

<400>15

tttggcgcat gctttccggc ccgtgattat gaacagaaaa aagtgctgcg cctgacctat 60

<210>16

<211>58

<212>DNA

<213> design of primer (P7 d)

<400>16

ccacaattgc tttatgggct tcctgcggat aatactgggt cagataggtc aggcgcag 58

<210>17

<211>57

<212>DNA

<213> design of primer (P8 u)

<400>17

ataaagcaat tgtggattat tggtttatgc gccatggcgg cgttgttccg ccgtatt 57

<210>18

<211>58

<212>DNA

<213> design of primer (P8 d)

<400>18

ccatctgctg ccggcggcgg ttcatagcct ttgctttctt caaaatacgg cggaacaa 58

<210>19

<211>52

<212>DNA

<213> design of primer (P9 u)

<400>19

gccggcagca gatggtggta gtccggcacc gccgggtgac gatgaagccc gt 52

<210>20

<211>48

<212>DNA

<213> design of primer (P9 d)

<400>20

gccctcgaga tcttcggttt caccttcatc ttcacgggct tcatcgtc 48

<210>21

<211>20

<212>DNA

<213> design of primer (mHVu 1)

<400>21

gatgtgaagc ttcaggagtc 20

<210>22

<211>20

<212>DNA

<213> design of primer (mHVu 2)

<400>22

caggtgcagc tgaaggagtc 20

<210>23

<211>20

<212>DNA

<213> design of primer (mHVu 3)

<400>23

caggtgcagc tgaagcagtc 20

<210>24

<211>20

<212>DNA

<213> design of primer (mHVu 4)

<400>24

caggttactc tgaaagagtc 20

<210>25

<211>20

<212>DNA

<213> design of primer (mHVu 5)

<400>25

aaggtccagc tgcaacaatc 20

<210>26

<211>20

<212>DNA

<213> design of primer (mHVu 6)

<400>26

gaggtccagc tgcagcagtc20

<210>27

<211>20

<212>DNA

<213> design of primer (mHVu 7)

<400>27

caggtccaac tgcagcagcc 20

<210>28

<211>20

<212>DNA

<213> design of primer (mHVu 8)

<400>28

gaggtgaagc tggtggagtc 20

<210>29

<211>20

<212>DNA

<213> design of primer (mHVu 9)

<400>29

gaggtgaagc tggtggaatc 20

<210>30

<211>20

<212>DNA

<213> design of primer (mHVu 10)

<400>30

gatgtgaact tggaagtgtc 20

<210>31

<211>21

<212>DNA

<213> design of primer (mHVd 1)

<400>31

tgcagagaca gtgaccagag t 21

<210>32

<211>21

<212>DNA

<213> design of primer (mHVd 2)

<400>32

tgaggagact gtgagagtgg t 21

<210>33

<211>21

<212>DNA

<213> design of primer (mHVd 3)

<400>33

tgaggagacg gtgactgagg t 21

<210>34

<211>21

<212>DNA

<213> design of primer (9mHVd4)

<400>34

tgaggagacg gtgaccgtgg t 21

<210>35

<211>21

<212>DNA

<213> design of primer (mKVu 1)

<400>35

gatgttttga tgacccaaac t 21

<210>36

<211>21

<212>DNA

<213> design of primer (mKVu 2)

<400>36

gatattgtga tgacgcaggc t 21

<210>37

<211>18

<212>DNA

<213> design of primer (mKVu 3)

<400>37

gatattgtga taacccag 18

<210>38

<211>21

<212>DNA

<213> design of primer (mKVu 4)

<400>38

gacattgtgc tgacccaatc t 21

<210>39

<211>21

<212>DNA

<213> design of primer (mKVu 5)

<400>39

gacattgtga tgacccagtc t 21

<210>40

<211>21

<212>DNA

<213> design of primer (mKVu 6)

<400>40

gatattgtgc taactcagtc t 21

<210>41

<211>21

<212>DNA

<213> design of primer (mKVu 7)

<400>41

gatatccaga tgacacagac t 21

<210>42

<211>21

<212>DNA

<213> design of primer (mKVu 8)

<400>42

gacatccagc tgactcagtc t 21

<210>43

<211>21

<212>DNA

<213> design of primer (mKVu 9)

<400>43

caaattgttc tcacccagtc t 21

<210>44

<211>19

<212>DNA

<213> design primer (mKVd 1)

<400>44

ccgtttcagc tccagcttg 19

<210>45

<211>21

<212>DNA

<213> design primer (mKVd 2)

<400>45

ccgttttatt tccagcttgg t 21

<210>46

<211>20

<212>DNA

<213> design primer (mKVd 3)

<400>46

ccgttttatt tccaactttg 20

<210>47

<211>714

<212>DNA

<213>rscFv

<400>47

gaggtgaagc tggtggaatc tggcgctgag gtgaagaagc ctggctcctc ggtgaaggtc 60

tcctgcacca gcagcgaagt gaccttcagc agcttcgcta tcagctgggt gcgccaggcc 120

ccgggtcaag gcctggagtg gctgggtggt attagcccga tgtttggcac cccgaactat 180

gcacagaagt tccagggccg cgtcacgatt accgcggacc agagcacccg caccgcgtat 240

atggatctgc gcagcctgcg ctctgaggac acggccgtgt attactgtgc gcgcagcccg 300

agctatattt gcagcggcgg cacctgcgtg tttgatcatt ggggccaggg cacctcagtc 360

accgtctcct caggcggcgg cggcagcgat attgtgataa cccagccgtc ggtgtccaag 420

ggcttgcgcc agaccgccac actgacctgc actggtaaca gcaacaatgt gggcaaccaa 480

ggtgcagctt ggctgcagca gcaccagggc caccctccca aactgctgtc ctacaggaat 540

aacgatcgcc cctcaggtat ttcagagcgc ttttctgcat cccgctcagg taacacagcc 600

tccctgacca ttactggcct gcagcctgag gacgaggctg actattactg ctcaacctgg 660

gacagcagcc tcagtgctgt ggtgttcggc ggcacaaagt tggaaataaa acgg 714

<210>48

<211>1479

<212>DNA

<213> gD-rscFv fusion sequence

<400>48

cctcgttata attacaccga acgctggcat accaccggtc cgattccgag cccgtttgcc 60

gatggccgcg aacagccggt ggaagttcgt tatgcaacca gtgcagccgc ctgcgatatg 120

ctggccctga ttgccgatcc gcaggtgggt cgcaccctgt gggaagcagt gcgtcgtcat 180

gcacgcgcct ataatgcaac cgttatttgg tataaaatcg aaagtggctg tgcacgcccg 240

ctgtattata tggaatatac cgaatgtgaa ccgcgcaaac attttggtta ttgccgttat 300

cgtaccccgc cgttttggga tagctttctg gcaggctttg cctatccgac cgatgatgaa 360

ctgggtctga ttatggccgc accggcccgt ctggtggaag gccaatatcg tcgcgcactg 420

tatattgatg gcaccgttgc atataccgat tttatggtta gtctgccggc cggcgattgc 480

tggtttagta aactgggtgc cgcacgtggt tatacctttg gcgcatgctt tccggcccgt 540

gattatgaac agaaaaaagt gctgcgcctg acctatctga cccagtatta tccgcaggaa 600

gcccataaag caattgtgga ttattggttt atgcgccatg gcggcgttgt tccgccgtat 660

tttgaagaaa gcaaaggcta tgaaccgccg ccggcagcag atggtggtag tccggcaccg 720

ccgggtgacg atgaagcccg tgaagatgaa ggtgaaaccg aagatgaggt gaagctggtg 780

gaatctggcg ctgaggtgaa gaagcctggc tcctcggtga aggtctcctg caccagcagc 840

gaagtgacct tcagcagctt cgctatcagc tgggtgcgcc aggccccggg tcaaggcctg 900

gagtggctgg gtggtattag cccgatgttt ggcaccccga actatgcaca gaagttccag 960

ggccgcgtca cgattaccgc ggaccagagc acccgcaccg cgtatatgga tctgcgcagc 1020

ctgcgctctg aggacacggc cgtgtattac tgtgcgcgca gcccgagcta tatttgcagc 1080

ggcggcacct gcgtgtttga tcattggggc cagggcacct cagtcaccgt ctcctcaggc 1140

ggcggcggca gcgatattgt gataacccag ccgtcggtgt ccaagggctt gcgccagacc 1200

gccacactga cctgcactgg taacagcaac aatgtgggca accaaggtgc agcttggctg 1260

cagcagcacc agggccaccc tcccaaactg ctgtcctaca ggaataacga tcgcccctca 1320

ggtatttcag agcgcttttc tgcatcccgc tcaggtaaca cagcctccct gaccattact 1380

ggcctgcagc ctgaggacga ggctgactat tactgctcaa cctgggacag cagcctcagt 1440

gctgtggtgt tcggcggcac aaagttggaa ataaaacgg 1479

Claims

1. A recombinant bovine herpes virus type I protein comprising a bovine herpes virus type I antigen and a bovine herpes virus type I single chain antibody;

the bovine herpes virus type I antigen is a gD antigen containing a major antigenic determinant;

the bovine herpes virus type I single-chain antibody is scFv.

2. A method of producing a recombinant bovine herpes virus type I protein according to claim 1, comprising the steps of:

(1) searching gD antigen containing main antigenic determinant in the bovine herpes virus I;

(2) optimizing codons of the bovine herpes virus I gD antigen gene, synthesizing a nucleic acid sequence and expressing the nucleic acid sequence;

(3) constructing a single-chain antibody aiming at the type I bovine herpes virus gD antigen, and calling the gene of the single-chain antibody scFv;

(4) recombining and expressing an optimized gene of the gD antigen of the bovine herpes virus type I and a gene of a single-chain antibody scFv aiming at the gD antigen of the bovine herpes virus type I in an induction manner; after induction, the culture solution is centrifuged at 5000rpm at 4 ℃ for 20min to collect thalli;

3. The process according to claim 2, wherein the steps (2) and (4) are carried out in an E.coli system.

4. The process according to claim 2, wherein the expression in steps (2) and (4) is carried out at an induction temperature of 20 ℃,25 ℃, 28 ℃ or 37 ℃, an induction rotation speed of 250rpm, and an IPTG concentration of 0.5 mM.

5. The process according to claim 2, wherein the induction temperature for the expression in steps (2) and (4) is 25 ℃ or 37 ℃.

6. The process according to claim 2, wherein the induction temperature for the expression in steps (2) and (4) is 25 ℃.

7. The process according to claim 2, wherein the purification of step (5) is selected from ion exchange chromatography, gel filtration chromatography or affinity chromatography.

8. The recombinant bovine herpes virus I protein as claimed in claim 2, wherein the purification method of step (5) is affinity chromatography.

9. The use of the recombinant bovine herpes virus type I protein of claim 1 in the preparation of a bovine herpes virus type I test kit.