CN110885378A - Peste des petits ruminants recombinant fusion protein, preparation method and application thereof - Google Patents

Peste des petits ruminants recombinant fusion protein, preparation method and application thereof Download PDF

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CN110885378A
CN110885378A CN201911286035.5A CN201911286035A CN110885378A CN 110885378 A CN110885378 A CN 110885378A CN 201911286035 A CN201911286035 A CN 201911286035A CN 110885378 A CN110885378 A CN 110885378A
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peste des
des petits
petits ruminants
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fusion protein
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李晓光
刘毅
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Hangzhou Billion Mino Biological Technology Co Ltd
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Hangzhou Billion Mino Biological Technology Co Ltd
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Abstract

The invention discloses a peste des petits ruminants recombinant fusion protein, a preparation method and application thereof, relating to the field of biomedicine, and comprising peste des petits ruminants antigen and peste des petits ruminants single-chain antibody; a method for preparing recombinant fusion protein of Peste des petits ruminants comprises the following steps of (1) searching N (425-525aa) antigen containing main antigenic determinant in Peste des petits ruminants; (2) optimizing the codon of the antigen gene of the peste des petits ruminants N (425-525aa), synthesizing a nucleic acid sequence and expressing the nucleic acid sequence; (3) constructing a single-chain antibody aiming at the peste des petits ruminants N (13-20aa) antigen, and calling a gene of the single-chain antibody scFv; (4) recombining and optimizing a peste des petits ruminants N (425-525aa) antigen gene and a single-chain antibody scFv gene aiming at the peste des petits ruminants N (13-20aa) antigen, and performing fusion expression; (5) purifying and renaturing the peste des petits ruminants recombinant fusion 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

Peste des petits ruminants recombinant fusion protein, preparation method and application thereof
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a peste des petits ruminants recombinant protein.
Background
Peste des petits ruminants (PPR) is commonly called sheep plague, also called false plague of Peste des petits ruminants, pneumonitis and stomatitis pneumonitis complex disease, and is an acute viral infectious disease caused by Peste des petits ruminants virus. PPR was discovered in india in 1978, and was outbreaked in the tibetan region of china in 2007. PPR is gaining increasing attention due to its widespread dissemination, impact on the economy and its important role in global cattle plague elimination activities.
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 raw materials for antibodies and antigens against Peste des petits ruminants to be used for producing diagnostic reagents is imminent, and to some extent, the development of raw materials is also bound to be the primary solution for reducing the spread of Peste des petits ruminants.
Disclosure of Invention
The invention provides a peste des petits ruminants recombinant fusion protein, which comprises a peste des petits ruminants antigen and a peste des petits ruminants single-chain antibody.
Further, the Peste des petits ruminants antigen is an N (425-525aa) antigen containing a main antigenic determinant, and the Peste des petits ruminants single-chain antibody is scFv.
The invention also provides a preparation method of the peste des petits ruminants recombinant protein, which comprises the following preparation steps:
(1) search for N (425-525aa) antigen containing major antigenic determinant in Peste des petits ruminants
From NCBI (national center for biotechnology information) N antigens were found that peste des petits contain major antigenic determinants.
(2) Optimizing the codon of the antigen of Peste des petits ruminants N (425-525aa), synthesizing the nucleic acid sequence and expressing
When expressing proteins in Escherichia coli, the frequencies of different codon usage are greatly different, in order to maximize the expression amount of recombinant proteins, the gene sequence of the Peste des petits ruminants N (425-525aa) antigen needs to be subjected to codon optimization, and then the optimized nucleic acid sequence is synthesized and expressed.
(3) Constructing single-chain antibody aiming at peste des petits ruminants N antigen, and calling gene of single-chain antibody scFv
Immunizing a mouse with the purified N antigen to obtain a high-activity monoclonal antibody, and then regulating the gene of the single-chain antibody scFv.
(4) Recombining and expressing the optimized peste des petits ruminants N antigen gene and the single-chain antibody scFv gene aiming at the peste des petits ruminants N antigen, connecting the optimized peste des petits ruminants N antigen (425-525aa) and the single-chain antibody scFv gene aiming at the peste des petits ruminants N antigen (capable of aiming at the part 13-20aa of the combined N antigen protein) by a recursive PCR technology, and inserting the gene segment of the recombinant protein into an expression vector for expression after sequencing and analyzing the correct sequence.
(5) Purifying and renaturing the peste des petits ruminants 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 0.1 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 method for expressing the peste des petits ruminants antigen and the single-chain antibody in an escherichia coli system by using a genetic engineering recombination technology has the advantages of short production period, high yield, low cost and the like.
The peste des petits ruminants recombinant protein can be used as a part of a peste des petits ruminants immunodiagnosis kit.
The invention has the following advantages and beneficial effects:
(1) single chain antibody scFv was first developed against the major antigen N of peste des petits ruminants.
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 antigen epitope of Peste des petits ruminants and scFv recombinant protein aiming at N antigen are developed for the first time at home and abroad.
Because no antibody is generated in the early stage of infection of the peste des petits ruminants, the corresponding peste des petits ruminants antibody cannot be detected in the period of time, so that missed detection is caused, in order to solve the problem, the detection of the peste des ruminants antibody and the detection of the antigen are increased, and the development of the antibody aiming at the N antigen of the peste des ruminants becomes a key point.
(3) The recombinant protein which simultaneously combines the peste des petits ruminants antibody and the antigen and has high activity, high sensitivity and high specificity is prepared.
After the main antigen epitope and the antibody aiming at the N antigen of the peste des petits ruminants are developed, the screened high-efficiency antigen epitope (425 and 525aa of the N antigen of the peste des petits ruminants) and the single-chain antibody scFv (13-20aa) aiming at the N antigen of the peste des petits ruminants are recombined by utilizing a genetic engineering technology to prepare the recombinant protein which has high activity, high sensitivity and high specificity and simultaneously combines the antibody and the antigen of the peste des petits ruminants, 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 for developing the diagnostic reagent of the peste des petits ruminants.
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 Peste des petits ruminants N antigen Gene and expression vector construction
Search for the N (425-525aa) antigen gene GenBank from NCBI: FJ905304.1, protein sequence as follows:
GGGESSAPATREGVKAAIPNGSEERDRKQTRPGRPRGETPGQLLLEIMPEDEVSRESGQNPREAQRSAEALFRLQAMAKILEDQEEGEDNNQVYNDKDLLG
the sequence after codon optimization is as follows:
GGCGGCGGCGAATCCTCCGCGCCGGCGACCCGTGAAGGCGTGAAAGCGGCGATTCCGAATGGCTCCGAAGAACGTGATCGTAAACAGACCCGTCCGGGCCGTCCGCGTGGCGAAACCCCGGGCCAGCTGCTGCTGGAAATTATGCCGGAAGATGAAGTGTCCCGTGAATCCGGCCAGAATCCGCGTGAAGCGCAGCGTTCCGCGGAAGCGCTGTTTCGTCTGCAGGCGATGGCGAAAATTCTGGAAGATCAGGAAGAAGGCGAAGATAATAATCAGGTGTATAATGATAAAGATCTGCTGGGC
primers were designed as follows:
P1u：GCCCATATGGGCGGCGGCGAATCCTCCGCGCCGGCGACCCGTGAAGGCGTGAAAGCGGC
P1d：ATCACGTTCTTCGGAGCCATTCGGAATCGCCGCTTTCACGCCT
P2u：TCCGAAGAACGTGATCGTAAACAGACCCGTCCGGGCCGTCCGCGTGGCGAAACC
P2d：TTCATCTTCCGGCATAATTTCCAGCAGCAGCTGGCCCGGGGTTTCGCCACGCGGACGG
P3u：ATGCCGGAAGATGAAGTGTCCCGTGAATCCGGCCAGAATCCGCGTGAAGCGCA
P3d：CGCCATCGCCTGCAGACGAAACAGCGCTTCCGCGGAACGCTGCGCTTCACGCGGA
P4u：CGCCATCGCCTGCAGACGAAACAGCGCTTCCGCGGAACGCTGCGCTTCACGCGGA
P4d：GCCCTCGAGGCCCAGCAGATCTTTATCATTATACACCTGATTATTATCTTCGCCTTCTTC
the experimental method is as follows:
mixing the primers P1u, P1d, P2u and P2d, and carrying out PCR reaction, wherein the obtained product is named as W1; mixing the primers P3u, P3d, P4u and P4d, and carrying out PCR reaction, wherein the obtained product is named as W2; then mixing the two products W1 and W2 as a template, and carrying out PCR reaction by using primers P1u and P4d to obtain the optimized N (425-525aa) gene of the Peste des petits ruminants. 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 ℃, 15 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 the core protein Peste des petits ruminants N antigen
(1) Acquisition of monoclonal antibody (directed against antibody binding to N antigen protein 13-20aa only) cell line
a. Transforming the pET30a-N (1-525aa) vector to BL21 expression strain for expression and purification to obtain N antigen protein
b. Immunizing mice with the obtained N (1-525aa) antigen protein
c. Monoclonal antibody technology is used for screening antibody cell strains only aiming at 13-20aa of combined N (1-525aa) antigen protein
(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 μ g RNA as a reverse transcription template, oligo (d) T1 μ 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 4. mu.l of 5 Xbuffer, 1. mu.l of RNase inhibitor and 2. mu.l of dNTP (10mM each) in sequence, and mixing
Incubating at 37 ℃ for 5 min;
1. mu.l of AMV reverse transcriptase was added,
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.
PCR reaction system 25 u l, wherein cDNA 0.5 u l, upstream primer 0.25 u l downstream primer 0.25 u l, Taq enzyme 0.2 u l dNTP 2u l 10 x buffer 2.5 u l.
PCR (TOYOBO Co., Ltd., cat # 02510D1) under the conditions: 94 ℃,2 min X1 cycles; (98 ℃, 5 seconds, 55 ℃, 30 seconds, 68 ℃, 15 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:
CAGGTGCAGCTGAAGGAGTCTGGCGCTGAGGTGAAGAAGCCTGGCTCCTCGGTGAAGGTCTCCTGCACCAGCAGCGAAGTGACCTTCAGCAGCTTCGCTATCAGCTGGGTGCGCCAGGCCCCGGGTCAAGGCCTGGAGTGGCTGGGTGGTATTAGCCCGATGTTTGGCACCCCGAACTATGCACAGAAGTTCCAGGGCCGCGTCACGATTACCGCGGACCAGAGCACCCGCACCGCGTATATGGATCTGCGCAGCCTGCGCTCTGAGGACACGGCCGTGTATTACTGTGCGCGCAGCCCGAGCTATATTTGCAGCGGCGGCACCTGCGTGTTTGATCATTGGGGCCAGGGCACCACTCTCACAGTCTCCTCAGGCGGCGGCGGCAGCGATATTGTGATGACGCAGGCTCCGTCGGTGTCCAAGGGCTTGCGCCAGACCGCCACACTGACCTGCACTGGTAACAGCAACAATGTGGGCAACCAAGGTGCAGCTTGGCTGCAGCAGCACCAGGGCCACCCTCCCAAACTGCTGTCCTACAGGAATAACGATCGCCCCTCAGGTATTTCAGAGCGCTTTTCTGCATCCCGCTCAGGTAACACAGCCTCCCTGACCATTACTGGCCTGCAGCCTGAGGACGAGGCTGACTATTACTGCTCAACCTGGGACAGCAGCCTCAGTGCTGTGGTGTTCGGCGGCACCAAGCTGGAAATAAAACGG
example 3 recombination of a Gene containing the N antigen of Peste des petits ruminants (425-525aa) and a Single-chain antibody against the N antigen of Peste des petits ruminants (13-20aa), expression of the recombinant gene
(1) The gene containing the N antigen of Peste des petits ruminants (425-525aa) and the gene of the single-chain antibody against the N antigen of Peste des petits ruminants (13-20aa) were ligated together by recursive PCR, double-digested with NdeI and XhoI restriction enzymes (available from NEB), and inserted into pET30a (Novagen product, cat # 69909-3) vector treated with the same two digests. The N (425-525aa) -rscFv fusion sequence is as follows:
GGCGGCGGCGAATCCTCCGCGCCGGCGACCCGTGAAGGCGTGAAAGCGGCGATTCCGAATGGCTCCGAAGAACGTGATCGTAAACAGACCCGTCCGGGCCGTCCGCGTGGCGAAACCCCGGGCCAGCTGCTGCTGGAAATTATGCCGGAAGATGAAGTGTCCCGTGAATCCGGCCAGAATCCGCGTGAAGCGCAGCGTTCCGCGGAAGCGCTGTTTCGTCTGCAGGCGATGGCGAAAATTCTGGAAGATCAGGAAGAAGGCGAAGATAATAATCAGGTGTATAATGATAAAGATCTGCTGGGCCAGGTGCAGCTGAAGGAGTCTGGCGCTGAGGTGAAGAAGCCTGGCTCCTCGGTGAAGGTCTCCTGCACCAGCAGCGAAGTGACCTTCAGCAGCTTCGCTATCAGCTGGGTGCGCCAGGCCCCGGGTCAAGGCCTGGAGTGGCTGGGTGGTATTAGCCCGATGTTTGGCACCCCGAACTATGCACAGAAGTTCCAGGGCCGCGTCACGATTACCGCGGACCAGAGCACCCGCACCGCGTATATGGATCTGCGCAGCCTGCGCTCTGAGGACACGGCCGTGTATTACTGTGCGCGCAGCCCGAGCTATATTTGCAGCGGCGGCACCTGCGTGTTTGATCATTGGGGCCAGGGCACCACTCTCACAGTCTCCTCAGGCGGCGGCGGCAGCGATATTGTGATGACGCAGGCTCCGTCGGTGTCCAAGGGCTTGCGCCAGACCGCCACACTGACCTGCACTGGTAACAGCAACAATGTGGGCAACCAAGGTGCAGCTTGGCTGCAGCAGCACCAGGGCCACCCTCCCAAACTGCTGTCCTACAGGAATAACGATCGCCCCTCAGGTATTTCAGAGCGCTTTTCTGCATCCCGCTCAGGTAACACAGCCTCCCTGACCATTACTGGCCTGCAGCCTGAGGACGAGGCTGACTATTACTGCTCAACCTGGGACAGCAGCCTCAGTGCTGTGGTGTTCGGCGGCACCAAGCTGGAAATAAAACGG
(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 0.1mM 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 Peste des petits ruminants
The mycelia were resuspended in 50ml of inclusion body extract (20mM Tris-HCl, 0.5MUreapH 7.5, 0.5M NaCl, 2% TritonX-100) and then disrupted by sonication under conditions of 3s per sonication, 6s intervals, 180 times total, 12000rpm, and centrifugation at 4 ℃ to collect inclusion body precipitates. The inclusion bodies were disrupted with 50ml of loading Buffer Binding Buffer (50mM Tris, 8M Urea, 0.5M NaCl, 20mM imidazole pH8.0), purified on a column, and the objective protein was eluted with Elution Buffer Elution Buffer (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> Peste des petits ruminants recombinant fusion protein, and preparation method and application thereof
<130>2
<160>38
<170>PatentIn version 3.3
<210>1
<211>101
<212>PRT
<213> N (425-525aa) antigen gene GenBank: protein sequence of FJ905304.1
<400>1
Gly Gly Gly Glu Ser Ser Ala Pro Ala Thr Arg Glu Gly Val Lys Ala
1 5 10 15
Ala Ile Pro Asn Gly Ser Glu Glu Arg Asp Arg Lys Gln Thr Arg Pro
20 25 30
Gly Arg Pro Arg Gly Glu Thr Pro Gly Gln Leu Leu Leu Glu Ile Met
35 40 45
Pro Glu Asp Glu Val Ser Arg Glu Ser Gly Gln Asn Pro Arg Glu Ala
5055 60
Gln Arg Ser Ala Glu Ala Leu Phe Arg Leu Gln Ala Met Ala Lys Ile
65 70 75 80
Leu Glu Asp Gln Glu Glu Gly Glu Asp Asn Asn Gln Val Tyr Asn Asp
85 90 95
Lys Asp Leu Leu Gly
100
<210>2
<211>303
<212>PRT
<213> N (425-525aa) antigen gene GenBank: protein sequence of FJ905304.1 after codon optimization
<400>2
ggcggcggcg aatcctccgc gccggcgacc cgtgaaggcg tgaaagcggc gattccgaat 60
ggctccgaag aacgtgatcg taaacagacc cgtccgggcc gtccgcgtgg cgaaaccccg 120
ggccagctgc tgctggaaat tatgccggaa gatgaagtgt cccgtgaatc cggccagaat 180
ccgcgtgaag cgcagcgttc cgcggaagcg ctgtttcgtc tgcaggcgat ggcgaaaatt 240
ctggaagatc aggaagaagg cgaagataat aatcaggtgt ataatgataa agatctgctg 300
ggc 303
<210>3
<211>59
<212>DNA
<213> design of primer (P1 u)
<400>3
gcccatatgg gcggcggcga atcctccgcg ccggcgaccc gtgaaggcgt gaaagcggc 59
<210>4
<211>43
<212>DNA
<213> design of primer (P1 d)
<400>4
atcacgttct tcggagccat tcggaatcgc cgctttcacg cct 43
<210>5
<211>54
<212>DNA
<213> design of primer (P2 u)
<400>5
tccgaagaac gtgatcgtaa acagacccgt ccgggccgtc cgcgtggcga aacc 54
<210>6
<211>58
<212>DNA
<213> design of primer (P2 d)
<400>6
ttcatcttcc ggcataattt ccagcagcag ctggcccggg gtttcgccac gcggacgg 58
<210>7
<211>53
<212>DNA
<213> design of primer (P3 u)
<400>7
atgccggaag atgaagtgtc ccgtgaatcc ggccagaatc cgcgtgaagc gca 53
<210>8
<211>55
<212>DNA
<213> design of primer (P3 d)
<400>8
cgccatcgcc tgcagacgaa acagcgcttc cgcggaacgc tgcgcttcac gcgga 55
<210>9
<211>55
<212>DNA
<213> design of primer (P4 u)
<400>9
cgccatcgcc tgcagacgaa acagcgcttc cgcggaacgc tgcgcttcac gcgga 55
<210>10
<211>60
<212>DNA
<213> design of primer (P4 d)
<400>10
gccctcgagg cccagcagat ctttatcatt atacacctga ttattatctt cgccttcttc 60
<210>11
<211>20
<212>DNA
<213> design of primer (mHVu 1)
<400>11
gatgtgaagc ttcaggagtc 20
<210>12
<211>20
<212>DNA
<213> design of primer (mHVu 2)
<400>12
caggtgcagc tgaaggagtc 20
<210>13
<211>20
<212>DNA
<213> design of primer (mHVu 3)
<400>13
caggtgcagc tgaagcagtc 20
<210>14
<211>20
<212>DNA
<213> design of primer (mHVu 4)
<400>14
caggttactc tgaaagagtc 20
<210>15
<211>20
<212>DNA
<213> design of primer (mHVu 5)
<400>15
aaggtccagc tgcaacaatc 20
<210>16
<211>20
<212>DNA
<213> design of primer (mHVu 6)
<400>16
gaggtccagc tgcagcagtc 20
<210>17
<211>20
<212>DNA
<213> design of primer (mHVu 7)
<400>17
caggtccaac tgcagcagcc 20
<210>18
<211>20
<212>DNA
<213> design of primer (mHVu 8)
<400>18
gaggtgaagc tggtggagtc 20
<210>19
<211>20
<212>DNA
<213> design of primer (mHVu 9)
<400>19
gaggtgaagc tggtggaatc 20
<210>20
<211>20
<212>DNA
<213> design of primer (mHVu 10)
<400>20
gatgtgaact tggaagtgtc 20
<210>21
<211>21
<212>DNA
<213> design of primer (mHVd 1)
<400>21
tgcagagaca gtgaccagag t 21
<210>22
<211>21
<212>DNA
<213> design of primer (mHVd 2)
<400>22
tgaggagact gtgagagtgg t 21
<210>23
<211>21
<212>DNA
<213> design of primer (mHVd 3)
<400>23
tgaggagacg gtgactgagg t 21
<210>24
<211>21
<212>DNA
<213> design of primer (mHVd 4)
<400>24
tgaggagacg gtgaccgtgg t 21
<210>25
<211>21
<212>DNA
<213> design of primer (mKVu 1)
<400>25
gatgttttga tgacccaaac t 21
<210>26
<211>21
<212>DNA
<213> design of primer (mKVu 2)
<400>26
gatattgtga tgacgcaggc t 21
<210>27
<211>18
<212>DNA
<213> design of primer (mKVu 3)
<400>27
gatattgtga taacccag18
<210>28
<211>21
<212>DNA
<213> design of primer (mKVu 4)
<400>28
gacattgtgc tgacccaatc t 21
<210>29
<211>21
<212>DNA
<213> design of primer (mKVu 5)
<400>29
gacattgtga tgacccagtc t 21
<210>30
<211>21
<212>DNA
<213> design of primer (mKVu 6)
<400>30
gatattgtgc taactcagtc t 21
<210>31
<211>21
<212>DNA
<213> design of primer (mKVu 7)
<400>31
gatatccaga tgacacagac t 21
<210>32
<211>21
<212>DNA
<213> design of primer (mKVu 8)
<400>32
gacatccagc tgactcagtc t 21
<210>33
<211>21
<212>DNA
<213> design of primer (mKVu 9)
<400>33
caaattgttc tcacccagtc t 21
<210>34
<211>19
<212>DNA
<213> design primer (mKVd 1)
<400>34
ccgtttcagc tccagcttg 19
<210>35
<211>21
<212>DNA
<213> design primer (mKVd 2)
<400>35
ccgttttatt tccagcttgg t 21
<210>36
<211>20
<212>DNA
<213> design primer (mKVd 3)
<400>36
ccgttttatt tccaactttg 20
<210>37
<211>717
<212>DNA
<213>rscFv
<400>37
caggtgcagc tgaaggagtc 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 caccactctc 360
acagtctcct caggcggcgg cggcagcgat attgtgatga cgcaggctcc gtcggtgtcc 420
aagggcttgc gccagaccgc cacactgacc tgcactggta acagcaacaa tgtgggcaac 480
caaggtgcag cttggctgca gcagcaccag ggccaccctc ccaaactgct gtcctacagg 540
aataacgatc gcccctcagg tatttcagag cgcttttctg catcccgctc aggtaacaca 600
gcctccctga ccattactgg cctgcagcct gaggacgagg ctgactatta ctgctcaacc 660
tgggacagca gcctcagtgc tgtggtgttc ggcggcacca agctggaaat aaaacgg 717
<210>38
<211>1020
<212>DNA
<213> N (425-525aa) -rscFv fusion sequences
<400>38
ggcggcggcg aatcctccgc gccggcgacc cgtgaaggcg tgaaagcggc gattccgaat 60
ggctccgaag aacgtgatcg taaacagacc cgtccgggcc gtccgcgtgg cgaaaccccg 120
ggccagctgc tgctggaaat tatgccggaa gatgaagtgt cccgtgaatc cggccagaat 180
ccgcgtgaag cgcagcgttc cgcggaagcg ctgtttcgtc tgcaggcgat ggcgaaaatt 240
ctggaagatc aggaagaagg cgaagataat aatcaggtgt ataatgataa agatctgctg 300
ggccaggtgc agctgaagga gtctggcgct gaggtgaaga agcctggctc ctcggtgaag 360
gtctcctgca ccagcagcga agtgaccttc agcagcttcg ctatcagctg ggtgcgccag 420
gccccgggtc aaggcctgga gtggctgggt ggtattagcc cgatgtttgg caccccgaac 480
tatgcacaga agttccaggg ccgcgtcacg attaccgcgg accagagcac ccgcaccgcg 540
tatatggatc tgcgcagcct gcgctctgag gacacggccg tgtattactg tgcgcgcagc 600
ccgagctata tttgcagcgg cggcacctgc gtgtttgatc attggggcca gggcaccact 660
ctcacagtct cctcaggcgg cggcggcagc gatattgtga tgacgcaggc tccgtcggtg 720
tccaagggct tgcgccagac cgccacactg acctgcactg gtaacagcaa caatgtgggc 780
aaccaaggtg cagcttggct gcagcagcac cagggccacc ctcccaaact gctgtcctac 840
aggaataacg atcgcccctc aggtatttca gagcgctttt ctgcatcccg ctcaggtaac 900
acagcctccc tgaccattac tggcctgcag cctgaggacg aggctgacta ttactgctca 960
acctgggaca gcagcctcag tgctgtggtg ttcggcggca ccaagctgga aataaaacgg 1020

Claims (9)

1. A peste des petits ruminants recombinant fusion protein is characterized by comprising peste des petits ruminants antigen and peste des petits ruminants single-chain antibody:
the peste des petits ruminants antigen is an N (425-525aa) antigen containing a main antigenic determinant;
the peste des petits ruminants single-chain antibody is scFv.
2. A method for preparing the peste des petits ruminants recombinant fusion protein based on the claim 1 is characterized by comprising the following steps:
(1) searching N (425-;
(2) optimizing the codon of the antigen gene of the peste des petits ruminants N (425-525aa), synthesizing a nucleic acid sequence and expressing the nucleic acid sequence;
(3) constructing a single-chain antibody aiming at the peste des petits ruminants N (13-20aa) antigen, and calling a gene of the single-chain antibody scFv;
(4) recombining and optimizing a peste des petits ruminants N (425-525aa) antigen gene and a single-chain antibody scFv gene aiming at the peste des petits ruminants N (13-20aa) antigen, and performing fusion expression;
(5) and (3) crushing the bacteria after induction expression by adopting an ultrasonic mode, setting the crushing condition as 200w, carrying out ultrasonic treatment for 6s every time at an interval of 3s for 180 times, and purifying and renaturing the peste des petits ruminants recombinant fusion protein.
3. The method for preparing recombinant fusion protein of Peste des petits ruminants according to claim 2, wherein the steps (2) and (4) are expressed in an Escherichia coli system.
4. The method for preparing recombinant fusion protein of Peste des petits ruminants according to claim 2, wherein the induction temperature in the expression of the step (2) and the step (4) is 20 ℃, 25 ℃, 28 ℃ or 37 ℃, the induction speed is 250rpm, and the concentration of induced IPTG is 0.1 mM.
5. The method for preparing recombinant Peste des petits ruminants fusion protein according to claim 2, wherein the induction temperature is 25 ℃ or 37 ℃.
6. The method for preparing recombinant Peste des petits ruminants fusion protein according to claim 2, wherein the induction temperature is 25 ℃.
7. The method for preparing recombinant fusion protein of Peste des petits ruminants according to claim 2, wherein the purification mode of the step (5) is selected from ion exchange chromatography, gel filtration chromatography or affinity chromatography.
8. The recombinant Peste des petits ruminants fusion protein and the preparation method thereof according to claim 2, wherein the purification mode of the step (5) is affinity chromatography.
9. The application of the peste des petits ruminants recombinant fusion protein is based on claim 1, and is characterized in that the recombinant protein is used for preparing a peste des petits ruminants detection kit.
CN201911286035.5A 2018-12-26 2019-12-13 Peste des petits ruminants recombinant fusion protein, preparation method and application thereof Pending CN110885378A (en)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1285513A (en) * 1999-08-19 2001-02-28 协和梅迪克斯株式会社 Method for testing or determining HCV core antigen and the agent for same
US20060127952A1 (en) * 2004-11-05 2006-06-15 Republic Of Korea (Management: Ministry Of Agriculture & Forestry, Ntl. Veterinary Research) Rapid diagnostic methods of Peste des Petits Ruminants using recombinant nucleocapsid protein expressed in insect cells and monoclonal antibody
WO2009079259A2 (en) * 2007-12-06 2009-06-25 Dana-Farber Cancer Institute, Inc. Antibodies against influenza virus and methods of use thereof
WO2011041691A1 (en) * 2009-10-02 2011-04-07 University Of Iowa Research Foundation Methods of improving vaccine immunogenicity
CN104558108A (en) * 2013-10-17 2015-04-29 上海市农业科学院 Linear epitope minimum motif peptides of PPRV (peste des petits ruminants virus) N (nucleocapsid) protein
CN105968175A (en) * 2016-04-14 2016-09-28 中国农业科学院哈尔滨兽医研究所 Recombinant peste des petits ruminants virus N protein and expression method and application thereof
CN107586783A (en) * 2016-07-06 2018-01-16 华中农业大学 Anti- PPR virus N protein monoclonal antibody and its application
CN108059684A (en) * 2017-12-25 2018-05-22 杭州亿米诺生物科技有限公司 A kind of bovine viral diarrhoea recombinant protein, its preparation method and application

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1285513A (en) * 1999-08-19 2001-02-28 协和梅迪克斯株式会社 Method for testing or determining HCV core antigen and the agent for same
US20060127952A1 (en) * 2004-11-05 2006-06-15 Republic Of Korea (Management: Ministry Of Agriculture & Forestry, Ntl. Veterinary Research) Rapid diagnostic methods of Peste des Petits Ruminants using recombinant nucleocapsid protein expressed in insect cells and monoclonal antibody
WO2009079259A2 (en) * 2007-12-06 2009-06-25 Dana-Farber Cancer Institute, Inc. Antibodies against influenza virus and methods of use thereof
WO2011041691A1 (en) * 2009-10-02 2011-04-07 University Of Iowa Research Foundation Methods of improving vaccine immunogenicity
CN104558108A (en) * 2013-10-17 2015-04-29 上海市农业科学院 Linear epitope minimum motif peptides of PPRV (peste des petits ruminants virus) N (nucleocapsid) protein
CN105968175A (en) * 2016-04-14 2016-09-28 中国农业科学院哈尔滨兽医研究所 Recombinant peste des petits ruminants virus N protein and expression method and application thereof
CN107586783A (en) * 2016-07-06 2018-01-16 华中农业大学 Anti- PPR virus N protein monoclonal antibody and its application
CN108059684A (en) * 2017-12-25 2018-05-22 杭州亿米诺生物科技有限公司 A kind of bovine viral diarrhoea recombinant protein, its preparation method and application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LAINE D. ET AL.: ""NCAP_PPRV，Accession NO：Q08823"", 《UNIPROT》 *
R. P. SINGH ET AL.: ""A sandwich-ELISA for the diagnosis of Peste des petits ruminants (PPR) infection in small ruminants using anti-nucleocapsid protein monoclonal antibody"", 《ARCHIVES OF VIROLOGY》 *
龙云凤 等: "小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用", 《畜牧兽医学报》 *

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