CN108178787A - PORF131 recombinant proteins and its preparation method and application - Google Patents

PORF131 recombinant proteins and its preparation method and application Download PDF

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CN108178787A
CN108178787A CN201711461888.9A CN201711461888A CN108178787A CN 108178787 A CN108178787 A CN 108178787A CN 201711461888 A CN201711461888 A CN 201711461888A CN 108178787 A CN108178787 A CN 108178787A
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porf131
recombinant proteins
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gly
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CN108178787B (en
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刘振兴
曹俊明
马艳平
梁志凌
马江耀
柯浩
郝乐
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Institute of Animal Health of Guangdong Academy of Agricultural Sciences
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

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Abstract

The invention discloses pORF131 recombinant proteins and its preparation method and application, the amino acid sequence shown in SEQ ID NO.7-SEQ ID NO.10 is formed by connecting pORF131 recombinant proteins by connexon successively, and the connexon is small 4-7 molecular weight, polarity and hydrophilic Amino acid profile.It is shown through ELISA, pORF131 recombinant proteins of the present invention can be used as envelope antigen, the DNA vaccination that 3 ORF131 complete encoding sequences of CyHV structure can be detected well is immunized the serum specific antibody generated after fancy carp and diagnoses whether fancy carp infects 3 type of carp herpesviral.

Description

PORF131 recombinant proteins and its preparation method and application
Technical field
The present invention relates to antigen-antibody fields, are specifically related to pORF131 recombinant proteins and its preparation method and application.
Background technology
3 type of carp herpesviral (Cyprinid herpesvirus 3, CyHV-3) is also known as Koi herpesvirus (Koi Herpesvirus, KHV), belong to herpesviral mesh (Herpesvirales), different herpetoviridae (Alloherpesviridae), carp Herpesvirus (Cyprinivirus) is a kind of double-stranded linear DNA virus, has cyst membrane Structure.The virus serious threat carp (Cyprinus carpio) and its fancy breed：Fancy carp, lethality are up to 80%-100%. 3 type genome about 295kb of carp herpesviral encodes 156 different ORF (Aoki etc., 2007).Envelope protein is located at virus most Outer layer, in virus with playing an important role and establishing detection method in the identification of host cell, interaction and phagocytic process With the important target molecule of means of prevention (Yi etc., 2014；Fuchs etc., 2014).CyHV-3 ORF131 belong to malicious envelope protein coding Gene, the gene include 2 intron structures (Liu Zhenxing, 2015), complete encoding sequence (complete CDS) overall length 1287bp, coding albumen include 428 amino acid.
CyHV-3 ORF131 are related corresponding up for further developing it.
Invention content
An object of the present invention is to provide new pORF131 recombinant proteins, can be used for detecting CyHV-3 ORF131 The serum specific antibody generated after fancy carp is immunized in the DNA vaccination of complete encoding sequence structure.
Realize that the technical solution of above-mentioned purpose is as follows.
PORF131 recombinant proteins, successively the amino acid sequence as shown in SEQ ID NO.7-SEQ ID NO.10 pass through company It connects son to be formed by connecting, the connexon is small 4-7 molecular weight, polarity and hydrophilic Amino acid profile.
The connexon composition is GGGGS.
A kind of expressing gene for encoding above-mentioned pORF131 recombinant proteins, sequence, including a：Its nucleotide sequence such as SEQ Shown in ID NO.6；b：With the nucleotide sequence coded mutually homotactic protein of a, but with a's due to the degeneracy of genetic code The different sequence of nucleotide sequence；C:Nucleotide sequence shown in above-mentioned a or b is carried out the substitutions of one or more bases, missing, Add the nucleotide sequence of modification.
The preparation method of above-mentioned pORF131 recombinant proteins, includes the following steps：
The expressing gene of the above-mentioned coding pORF131 recombinant proteins is inserted into pET32a by HindIII/XhoI double digestions (+) carrier, construction recombination plasmid pET32a-modORF131；
It is transferred to DH5 α again, PCR screening positive transformants bacterial strains extract plasmid after positive strain sequencing identification, are transferred to respectively BL21 (DE3) expresses bacterial strain；
BL21 (DE3) the expression bacterial strains of picking conversion pET32a-modORF131, are inoculated in the LB culture mediums of Amp resistances Overnight incubation is inoculated in LB fresh cultures, add IPTG induced expressions, purifying to get.
Encode the sequence such as SEQ ID NO.6 of the expressing gene of the pORF131 recombinant proteins.
It is detected in fancy carp serum for the specific antibody of CyHV-3pORF131 it is a further object of the present invention to provide a kind of Kit.
Specific technical solution is as follows：
The kit of the serum specific antibody of fancy carp is detected, includes the pORF131 recombinant proteins.
The kit is ELISA kit in one of the embodiments, including the useful pORF131 recombinations egg White coated ELISA Plate.
The polyclonal antibody of the anti-fancy carp IgM of mouse is further included in one of the embodiments,.
The present invention by inventor intercepted epitope advantage section in CyHV-3pORF131 whole protein sequences into It has gone prokaryotic expression, has been shown through ELISA, envelope antigen can be used as by truncating the albumen (SEQ ID NO.5) of expression, can be well The serum specific antibody generated after fancy carp is immunized in DNA vaccination to detect CyHV-3 ORF131 complete encoding sequences structure, And whether diagnosis fancy carp infects 3 type of carp herpesviral.
Description of the drawings
Fig. 1 is the recombinant expression of pORF131, wherein, M:Protein Marker, 1：Pet32a (+) is not induced, and 2： Pet32a (+) induction, 3：Pet32a (+)-compORF131 is not induced, and 4：Pet32a (+)-compORF131 is induced, and 5： Pet32a (+)-modORF131 is not induced, and 6：Pet32a (+)-modORF131 is induced.
The Western blot identifications that Fig. 2 is recombinant expression pORF131, wherein M:Protein Marker, 1：pet32a (+)-modORF131 is induced.
Fig. 3 is purified for fancy carp serum IgM, M:Albumen Marker, 1：Do not purify fancy carp serum, 2：Washing percolation liquid, 3：It washes De- liquid.
Specific embodiment
Embodiment described below, only preferred embodiment of the invention, in practical operation, can select different as needed Separation condition.
First, materials and methods
The sequence analysis of 1.CyHV-3 ORF131 and optimization, synthesis
CyHV-3 ORF131 gene code viral envelope proteins, to ORF131 genes and protein sequence (GenBank No.KP004905, AJK93611) it is analyzed, predict epitope advantage section.Finally, according to the experience of inventor, pass through It repeatedly assesses, after the coded sequence (SEQ ID NO.5) for determining expression epitope advantage section, Song Jinwei intelligence company carries out close Numeral optimizes and sequent synthesis.
2.pET32a-compORF131, the construction and expressions of pET32a-modORF131 recombinant plasmids
Table 1ORF131 amplimers sequence (5 ' -3 ')
Table 1Sequence of ORF131gene amplification
RNA is extracted from the culture of CyHV-3HZ419 plants of infection CCB cells using Trizol reagents, with the RNA of extraction As template, reference1st Strand cDNA synthetic agent box (Takara) specification the first chains of reverse transcription cDNA.Using above-mentioned first chain cDNA as template, using Prime Star Max high-fidelity enzymes, with primer 131Hind3CF/ 131XholR (table 1) expands ORF131 complete encoding sequences.Response procedures use：94 DEG C of 5min pre-degenerations；94℃30s,59℃ 30s, 72 DEG C of 90s, 35 cycles；72 DEG C of 5min overall elongations, 4 DEG C of preservations.The coding for antigens epitope Predominance Area synthesized with Jin Weizhi The ORF131 (SEQ ID NO.6) of section is template, with primer 131Hind3MF/131XholMR (table 1) amplification optimizations ORF131。
Above-mentioned ORF131 complete encoding sequences amplified production and the amplified production (SEQ ID NO.6) for optimizing segment is logical It crosses HindIII/XhoI double digestions and is inserted into pET32a (+) carrier respectively, the recombinant plasmid of structure is respectively designated as pET32a- CompORF131, pET32a-modORF131, are transferred to DH5 α, and PCR screening positive transformants bacterial strains carry after positive strain sequencing identification Plasmid is taken, is transferred to BL21 (DE3) expression bacterial strains respectively.Picking is transferred to BL21 (DE3) the expression bacterial strains of recombinant plasmid, is inoculated in Overnight incubation in the LB culture mediums of Amp resistances, then by 1:100 ratios are inoculated in 100mL LB fresh cultures, 37 DEG C, 220rpm/min, which is cultivated to bacterium solution OD values, reaches 0.4-0.5, adds in 0.8mM IPTG in 28 DEG C, induced expression.With reference to Novagen Pet sheet carries out protein expression and soluble analysis up to system handbook.The protein expressioning product under optimum condition of the expression is chosen, is used Novagen His Bind protein purification kits are purified, and purifying protein -80 DEG C of preservations after BCA kit quantifications obtain To pORF131 (pET32a-modORF131) expression albumen (SEQ ID NO.5).
3. recombinate the Western-blot analyses of pORF131
After purifying protein SDS-PAGE electrophoresis, through 100mA, 1.5h goes to pvdf membrane, 5% 37 DEG C of skimmed milk power confining liquid envelope PBST washes film 3 times after closing 2h, adds in 1:Anti- His labels mouse monoclonal antibody (the Jin Sirui biologies section of 1000 diluted HRP labels Skill Co., Ltd) 37 DEG C be incubated 2h, PBST is detected after washing film 5 times with Bio-Rad bioluminescent reagents box.
4. fancy carp serum specific antibody detects the foundation of ELISA method
The purifying of 4.1 fancy carp serum IgMs
Fancy carp serum is acquired, is mixed in equal volume with 4moL/L NaCl (pH8.3), 1mL is taken to add in 2moL/L NaCl (pH8.3) the rProteinG prepacked columns (Beijing Webster Bo Hui chromatographies Science and Technology Ltd.) after balancing, are stored at room temperature 15min, 2moL/L NaCl (pH8.3) wash 10 column beds, and 0.05moL/L Gly are eluted, collection eluent, and 1:50 add in Tris-HCl (pH8.0) pH is adjusted.Purified product carries out SDS-PAGE electrophoretic analysis.Band Qie Jiao Hou Songmeiji biotech firms are purified to carry out LC-MS/MS is analyzed.
The preparation of the polyclonal antibody of the anti-fancy carp IgM of 4.2 mouse
It using the carp IgM of above-mentioned purifying as mice immunized with antigen, is immunized 3 times, every minor tick 2 weeks, the 1st time antigen adds in equal volume Freund's complete adjuvant, latter 2 times plus isometric incomplete Freund's adjuvant, for 50 μ g/ only, immunization route is back to immunizing dose With subcutaneous abdomen multi-point injection.3 days booster immunizations before blood sampling are injected intraperitoneally with adjuvant antigen is not added with, and dosage is 50 μ g/.It plucks Except eyeball is taken a blood sample, separation serum uses rProtein A (Beijing Webster Bo Hui chromatographies Science and Technology Ltd.), purified mouse IgG ,- 80 DEG C of preservations.
5.ORF131 DNA vaccinations are immunized
With reference to the method for Liu Zhenxing (2015), Bgl II, III digestions of Hind are utilized after ORF131 complete encoding sequences are expanded PEGFP-N1 carriers are inserted into site, and average body is immunized as DNA vaccination intramuscular injection in the recombinant plasmid pEGFP-ORF131 of structure The fancy carp of weight 20g, immunizing dose is 3 μ g/ tails, while sets up pEGFP-N1 vector immunities group as negative control, after 3 weeks immune Acquire serum, -80 DEG C of preservations.
6. indirect EILSA methods detect immune serum antibody titer
Above-mentioned pORF131 (pET32a-modORF131) the expression albumen of purifying, 320 μ g/mL, 100 μ are diluted to CBS L/ holes coated elisa plate, 4 DEG C overnight, and PBST is washed 3 times；Add in confining liquid (1%BSA), 37 DEG C of closing 1.5h, PBST washings 3 It is secondary；Add in 1:300 diluted fancy carp serum (pass through fancy carp serum and the conduct pair that DNA vaccination is immunized including above-mentioned 5th point According to pEGFP-N1 vector immunities fancy carp serum) 100 μ L/ holes, 25 DEG C incubation 1.5h, PBST wash 5 times；Add in 100 μ L/ holes 1:The 3000 dilution anti-fancy carp polyclonal antibodies of mouse (above-mentioned 4.2 is preparation-obtained), 37 DEG C are incubated 2.5h, and PBST is washed 5 times；Add Enter 100 μ L/ holes 1:8000 dilution HRP label goat anti-mouse iggs (Beijing DingGuo ChangSheng Biology Technology Co., Ltd), 37 DEG C 2h is incubated, PBST is washed 5 times；Add in 100 μ L/ holes TMB working solutions, 37 DEG C of incubation 30min.50 μ L/ holes terminate liquids are added in, are detected OD450。
2nd, interpretation of result
1st, pORF131 sequence analyses and B cell epitope advantage section are predicted
CyHV-3HZ419 plants of ORF131 complete encoding sequence overall length 1287bp, coding albumen include 428 amino acid.It should Gene includes 1 signal peptide (Met1~Gly22) and 1 transmembrane segment (Phe393~Ile415).By grinding for inventor After studying carefully, 4 B cell epitope advantage sections of final determining pORF131：Cys20-Val140,Ser169-Tyr245,Thr259- Pro390,Phe414-Gln428。
Rare codon analysis shows that, low abundance codon (threshold in CyHV-3 ORF131 complete encoding sequences =10) there are 71 (E.coli Codon Usage Analyzer 2.1), to improve protein expression efficiency, above-mentioned 4 B cells Epitope advantage section (SEQ ID.NO.7-10) is connected using Gly-Gly-Gly-Gly-Ser (G4S) flexible section (connexon) (SEQ ID.NO.5) Song Jinwei intelligence company afterwards carries out codon optimization and sequence (SEQ according to Escherichia coli Preference codon ID.NO.6 it) synthesizes.Particular sequence is as follows.
SEQ ID NO.5：
CQGDDDISVSCDDVTGSVGKEVTLTCNISLQCPDCSIKKCKFRSPTDSVICEQELLNDSCEQSNSFTCRYTTTTAMT EKFSFFVQTKCGMKQTEFTVDISDITISFETPELVEAYVNDVKV(SEQ ID.NO.7)GGGGSSGRVMIPEEEFLIQTAGLTTIPPTTRPTPPTAATLPPIITINVADPTTTRPTTTFTLPPTTPTTPSLEQLAA SRAVY(SEQ ID.NO.8)
GGGGSTTPLPTTTTTPGTTTPPAPTPGLVVPLAQLREAFDPVYYSSLAPDTLPLPQPQVARIIAGPSTIRYKVTDPP PTHAPTVAMSTLPPSTTASTAPTTPTVPRSTLHWLNPAGFANQFHNCGSGSEALFKCSKGP(SEQ ID.NO.9)GGGGSFIKKRNAPVNYHPLQ(SEQ ID.NO.10)。
The codon optimised sequence of pORF131B cell epitope advantage sections
TGCCAGGGCGACGATGATATCAGCGTGAGTTGCGACGATGTTACCGGTAGCGTGGGCAAAGAGGTGACCCTGACCTG CAACATCAGCCTGCAGTGCCCGGATTGTAGCATTAAGAAATGCAAGTTTCGTAGTCCGACCGACAGCGTTATTTGCG AGCAGGAACTGCTGAACGATAGCTGCGAGCAGAGTAACAGCTTCACCTGCCGCTATACCACCACCACCGCCATGACC GAGAAATTCAGTTTCTTTGTGCAAACCAAATGCGGCATGAAACAGACCGAGTTCACCGTGGACATCAGTGACATCAC CATCAGCTTTGAAACCCCGGAACTGGTGGAGGCCTACGTGAATGACGTGAAGGTTGGCGGTGGCGGCAGTAGTGGTC GCGTTATGATCCCGGAAGAAGAGTTTCTGATCCAGACCGCCGGCTTAACCACAATTCCGCCTACCACCCGTCCGACC CCCCCTACAGCAGCAACCCTGCCGCCGATCATTACCATTAACGTGGCAGACCCGACCACCACCCGTCCGACAACCAC CTTCACCCTGCCGCCTACCACCCCTACCACACCGAGCCTGGAACAGCTGGCAGCAAGCCGTGCCGTTTATGGCGGTG GCGGTAGCACCACACCGCTGCCGACCACAACCACCACACCGGGTACCACAACACCGCCGGCCCCTACACCTGGTCTG GTTGTGCCTCTGGCCCAGCTGCGTGAGGCCTTTGACCCGGTTTACTATAGCAGCCTGGCCCCGGATACCTTACCGCT GCCTCAGCCTCAGGTGGCCCGCATTATCGCAGGCCCGAGCACAATTCGCTATAAGGTTACCGATCCGCCTCCGACCC ATGCACCTACCGTGGCAATGAGCACCTTACCGCCGAGTACCACCGCCAGCACAGCACCGACAACCCCGACAGTGCCG CGTAGCACCTTACACTGGCTGAATCCGGCCGGCTTTGCCAATCAGTTCCACAACTGCGGTAGCGGCAGCGAGGCCCT GTTTAAATGCAGCAAAGGCCCTGGTGGCGGCGGTAGCTTTATCAAGAAGCGCAACGCACCGGTGAATTATCACCCGC TGCAG(SEQ ID NO.6)。
2. the induced expression of recombinant protein
The fusion protein theoretical molecular weight of pet32a (+)-compORF131 and pet32a (+)-modORF131 expression is distinguished For 65.7kDa, 57.8kDa, using 0.8mM IPTG, 28 DEG C of induction 4h only pet32a (+)-modORF131 are attached in purpose band The nearly visible band (Fig. 1) significantly expressed, pet32a (+)-compORF131 have no the item significantly expressed near purpose band Band.Soluble analysis shows that albumen is expressed with inclusion bodies.Utilize Novagen His Bind protein purification kits Destination protein, the mouse monoclonal antibody Western-blot analyses through anti-His labels, hybridizes to apparent band at 57.8kD (Fig. 2).
3. recombinate applications of the pORF131 in ELISA method
The purifying of 3.1 carp serum IgMs and the preparation of the anti-carp polyclonal antibody of mouse
SDS-PAGE is shown in>Nearby there is apparent band in 70kDa, thus it is speculated that for fancy carp IgM heavy chains (Fig. 3), Mei Ji companies LC-MS/MS Mass Spectrometric Identifications confirm that the albumen is carp serum IgM heavy chain.With IgM points of 3 immune mouse of fancy carp of purifying, strengthen exempting from 3 days after epidemic disease, eyeball blood sampling is extractd, ELISA detects the Mouse Antisera potency for showing preparation>1:160,000.
The application of 3.2 detection of specific antibody ELISA methods
Using the anti-fancy carp IgM polyclonal antibodies of mouse prepared by this research as detection antibody, using the recombination pORF131 of purifying as packet By antigen, the indirect EILSA methods detection fancy carp immune serum antibody titer of foundation, the results showed that fancy carp serum is immunized in 5 tails OD450 for be respectively 0.532,0.522,0.511,0.488,0.482, pEGFP-N1 vector immunities serum (negative control) OD450 is≤0.220, P/N ＞ 2.1.Repeatedly, testing result is stablized.This method can evaluate specific antibody level.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, it is all considered to be the range of this specification record.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that those of ordinary skill in the art are come It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
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Cys Gln Gly Asp Asp Asp Ile Ser Val Ser Cys Asp Asp Val Thr Gly
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Pro Asp Cys Ser Ile Lys Lys Cys Lys Phe Arg Ser Pro Thr Asp Ser
35 40 45
Val Ile Cys Glu Gln Glu Leu Leu Asn Asp Ser Cys Glu Gln Ser Asn
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Ser Phe Thr Cys Arg Tyr Thr Thr Thr Thr Ala Met Thr Glu Lys Phe
65 70 75 80
Ser Phe Phe Val Gln Thr Lys Cys Gly Met Lys Gln Thr Glu Phe Thr
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Val Asp Ile Ser Asp Ile Thr Ile Ser Phe Glu Thr Pro Glu Leu Val
100 105 110
Glu Ala Tyr Val Asn Asp Val Lys Val Gly Gly Gly Gly Ser Ser Gly
115 120 125
Arg Val Met Ile Pro Glu Glu Glu Phe Leu Ile Gln Thr Ala Gly Leu
130 135 140
Thr Thr Ile Pro Pro Thr Thr Arg Pro Thr Pro Pro Thr Ala Ala Thr
145 150 155 160
Leu Pro Pro Ile Ile Thr Ile Asn Val Ala Asp Pro Thr Thr Thr Arg
165 170 175
Pro Thr Thr Thr Phe Thr Leu Pro Pro Thr Thr Pro Thr Thr Pro Ser
180 185 190
Leu Glu Gln Leu Ala Ala Ser Arg Ala Val Tyr Gly Gly Gly Gly Ser
195 200 205
Thr Thr Pro Leu Pro Thr Thr Thr Thr Thr Pro Gly Thr Thr Thr Pro
210 215 220
Pro Ala Pro Thr Pro Gly Leu Val Val Pro Leu Ala Gln Leu Arg Glu
225 230 235 240
Ala Phe Asp Pro Val Tyr Tyr Ser Ser Leu Ala Pro Asp Thr Leu Pro
245 250 255
Leu Pro Gln Pro Gln Val Ala Arg Ile Ile Ala Gly Pro Ser Thr Ile
260 265 270
Arg Tyr Lys Val Thr Asp Pro Pro Pro Thr His Ala Pro Thr Val Ala
275 280 285
Met Ser Thr Leu Pro Pro Ser Thr Thr Ala Ser Thr Ala Pro Thr Thr
290 295 300
Pro Thr Val Pro Arg Ser Thr Leu His Trp Leu Asn Pro Ala Gly Phe
305 310 315 320
Ala Asn Gln Phe His Asn Cys Gly Ser Gly Ser Glu Ala Leu Phe Lys
325 330 335
Cys Ser Lys Gly Pro Gly Gly Gly Gly Ser Phe Ile Lys Lys Arg Asn
340 345 350
Ala Pro Val Asn Tyr His Pro Leu Gln
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aagtttcgta gtccgaccga cagcgttatt tgcgagcagg aactgctgaa cgatagctgc 180
gagcagagta acagcttcac ctgccgctat accaccacca ccgccatgac cgagaaattc 240
agtttctttg tgcaaaccaa atgcggcatg aaacagaccg agttcaccgt ggacatcagt 300
gacatcacca tcagctttga aaccccggaa ctggtggagg cctacgtgaa tgacgtgaag 360
gttggcggtg gcggcagtag tggtcgcgtt atgatcccgg aagaagagtt tctgatccag 420
accgccggct taaccacaat tccgcctacc acccgtccga ccccccctac agcagcaacc 480
ctgccgccga tcattaccat taacgtggca gacccgacca ccacccgtcc gacaaccacc 540
ttcaccctgc cgcctaccac ccctaccaca ccgagcctgg aacagctggc agcaagccgt 600
gccgtttatg gcggtggcgg tagcaccaca ccgctgccga ccacaaccac cacaccgggt 660
accacaacac cgccggcccc tacacctggt ctggttgtgc ctctggccca gctgcgtgag 720
gcctttgacc cggtttacta tagcagcctg gccccggata ccttaccgct gcctcagcct 780
caggtggccc gcattatcgc aggcccgagc acaattcgct ataaggttac cgatccgcct 840
ccgacccatg cacctaccgt ggcaatgagc accttaccgc cgagtaccac cgccagcaca 900
gcaccgacaa ccccgacagt gccgcgtagc accttacact ggctgaatcc ggccggcttt 960
gccaatcagt tccacaactg cggtagcggc agcgaggccc tgtttaaatg cagcaaaggc 1020
cctggtggcg gcggtagctt tatcaagaag cgcaacgcac cggtgaatta tcacccgctg 1080
cag 1083
<210> 7
<211> 121
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 7
Cys Gln Gly Asp Asp Asp Ile Ser Val Ser Cys Asp Asp Val Thr Gly
1 5 10 15
Ser Val Gly Lys Glu Val Thr Leu Thr Cys Asn Ile Ser Leu Gln Cys
20 25 30
Pro Asp Cys Ser Ile Lys Lys Cys Lys Phe Arg Ser Pro Thr Asp Ser
35 40 45
Val Ile Cys Glu Gln Glu Leu Leu Asn Asp Ser Cys Glu Gln Ser Asn
50 55 60
Ser Phe Thr Cys Arg Tyr Thr Thr Thr Thr Ala Met Thr Glu Lys Phe
65 70 75 80
Ser Phe Phe Val Gln Thr Lys Cys Gly Met Lys Gln Thr Glu Phe Thr
85 90 95
Val Asp Ile Ser Asp Ile Thr Ile Ser Phe Glu Thr Pro Glu Leu Val
100 105 110
Glu Ala Tyr Val Asn Asp Val Lys Val
115 120
<210> 8
<211> 77
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 8
Ser Gly Arg Val Met Ile Pro Glu Glu Glu Phe Leu Ile Gln Thr Ala
1 5 10 15
Gly Leu Thr Thr Ile Pro Pro Thr Thr Arg Pro Thr Pro Pro Thr Ala
20 25 30
Ala Thr Leu Pro Pro Ile Ile Thr Ile Asn Val Ala Asp Pro Thr Thr
35 40 45
Thr Arg Pro Thr Thr Thr Phe Thr Leu Pro Pro Thr Thr Pro Thr Thr
50 55 60
Pro Ser Leu Glu Gln Leu Ala Ala Ser Arg Ala Val Tyr
65 70 75
<210> 9
<211> 133
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 9
Thr Thr Pro Leu Pro Thr Thr Thr Thr Thr Pro Gly Thr Thr Thr Pro
1 5 10 15
Pro Ala Pro Thr Pro Gly Leu Val Val Pro Leu Ala Gln Leu Arg Glu
20 25 30
Ala Phe Asp Pro Val Tyr Tyr Ser Ser Leu Ala Pro Asp Thr Leu Pro
35 40 45
Leu Pro Gln Pro Gln Val Ala Arg Ile Ile Ala Gly Pro Ser Thr Ile
50 55 60
Arg Tyr Lys Val Thr Asp Pro Pro Pro Thr His Ala Pro Thr Val Ala
65 70 75 80
Met Ser Thr Leu Pro Pro Ser Thr Thr Ala Ser Thr Ala Pro Thr Thr
85 90 95
Pro Thr Val Pro Arg Ser Thr Leu His Trp Leu Asn Pro Ala Gly Phe
100 105 110
Ala Asn Gln Phe His Asn Cys Gly Ser Gly Ser Glu Ala Leu Phe Lys
115 120 125
Cys Ser Lys Gly Pro
130
<210> 10
<211> 15
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 10
Phe Ile Lys Lys Arg Asn Ala Pro Val Asn Tyr His Pro Leu Gln
1 5 10 15

Claims (9)

1.pORF131 recombinant proteins, it is characterized in that, the amino acid sequence as shown in SEQ ID NO.7-SEQ ID NO.10 successively Row are formed by connecting by connexon, and the connexon is small 4-7 molecular weight, polarity and hydrophilic Amino acid profile.
2. recombinant protein according to claim 1, it is characterized in that, the connexon composition is Gly-Gly-Gly-Gly- Ser。
3. a kind of expressing gene for encoding pORF131 recombinant proteins described in claim 1, it is characterized in that, sequence, including a： Its nucleotide sequence is as shown in SEQ ID NO.6；b：With the nucleotide sequence coded mutually homotactic protein of a, but because of heredity The degeneracy of password and the sequence different from the nucleotide sequence of a；C:To nucleotide sequence shown in above-mentioned a or b carry out one or The nucleotide sequence that substitution, missing, the addition of multiple bases are modified.
4. the preparation method of pORF131 recombinant proteins described in claim 1, it is characterized in that, include the following steps：
The expressing gene of the coding pORF131 recombinant proteins is inserted by HindIII/XhoI double digestions described in claim 3 PET32a (+) carrier, construction recombination plasmid pET32a-modORF131；
It is transferred to DH5 α again, PCR screening positive transformants bacterial strains extract plasmid after positive strain sequencing identification, are transferred to BL21 respectively (DE3) bacterial strain is expressed；
BL21 (DE3) the expression bacterial strains of picking conversion pET32a-modORF131, are inoculated in the LB culture mediums of Amp resistances and cultivate Overnight, be inoculated in LB fresh cultures, add IPTG induced expressions, purifying to get.
5. preparation method according to claim 4, it is characterized in that, the expression of the coding pORF131 recombinant proteins The sequence of gene is as shown in SEQ ID NO.6.
6. claim 1-2 any one of them pORF131 recombinant proteins are preparing the serum specific antibody of detection fancy carp Application in kit.
7. the kit of the serum specific antibody of fancy carp is detected, it is characterized in that, include described in claims 1 or 2 PORF131 recombinant proteins.
8. the kit of the serum specific antibody of detection fancy carp according to claim 7, it is characterized in that, the kit For ELISA kit, including the coated ELISA Plate of pORF131 recombinant proteins described in useful claims 1 or 2.
9. the kit of the serum specific antibody of detection fancy carp according to claim 7 or 8, it is characterized in that, it further includes There is the polyclonal antibody of the anti-fancy carp IgM of mouse.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114958806A (en) * 2022-06-14 2022-08-30 中国水产科学研究院淡水渔业研究中心 Carp LPL1 recombinant protein and preparation method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450627A (en) * 2014-12-24 2015-03-25 广东省农业科学院动物卫生研究所 Monoclonal antibody of cyprinid herpesvirus III envelope protein ORF132, hybridoma cell strain and application of monoclonal antibody
CN107056898A (en) * 2017-02-13 2017-08-18 中国水产科学研究院珠江水产研究所 3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expressions albumen, antibody and its application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450627A (en) * 2014-12-24 2015-03-25 广东省农业科学院动物卫生研究所 Monoclonal antibody of cyprinid herpesvirus III envelope protein ORF132, hybridoma cell strain and application of monoclonal antibody
CN107056898A (en) * 2017-02-13 2017-08-18 中国水产科学研究院珠江水产研究所 3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expressions albumen, antibody and its application

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114958806A (en) * 2022-06-14 2022-08-30 中国水产科学研究院淡水渔业研究中心 Carp LPL1 recombinant protein and preparation method thereof

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