CN108047317A - PORF148 recombinant proteins and its preparation method and application - Google Patents

PORF148 recombinant proteins and its preparation method and application Download PDF

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CN108047317A
CN108047317A CN201810128920.XA CN201810128920A CN108047317A CN 108047317 A CN108047317 A CN 108047317A CN 201810128920 A CN201810128920 A CN 201810128920A CN 108047317 A CN108047317 A CN 108047317A
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porf148
recombinant proteins
seq
sequence
kit
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CN108047317B (en
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郝乐
梁志凌
马江耀
曹俊明
柯浩
马艳平
刘振兴
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Institute of Animal Health of Guangdong Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Abstract

The invention discloses pORF148 recombinant proteins and its preparation method and application, the amino acid sequence shown in SEQ ID NO.13-SEQ ID NO.16 is formed by connecting the pORF148 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, pORF148 recombinant proteins of the present invention can be used as envelope antigen, and the serum specific antibody generated after fancy carp is immunized in the DNA vaccination that can detect CyHV 3pORF148 complete encoding sequences structure well.

Description

PORF148 recombinant proteins and its preparation method and application
Technical field
The present invention relates to antigen-antibody fields, are specifically related to pORF148 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 (Cyprinuscarpio) 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), has determined at present therein 14 ORF coding envelope protein (Michel etc., 2010；Yi etc., 2014).Envelope protein is located at viral outermost layer, virus with Detection method and means of prevention are played an important role and established in the identification of host cell, interaction and phagocytic process Important target molecule (Yi etc., 2014；Fuchs etc., 2014).Therefore expression CyHV-3 envelope proteins antigen is with preparing anti-carp serum The antibody of IgM, which becomes, establishes detection method key issue urgently to be resolved hurrily.
The content of the invention
An object of the present invention is to provide new pORF148 recombinant proteins, can be used for detecting CyHV-3 The serum specific antibody generated after fancy carp is immunized in the DNA vaccination of pORF148 complete encoding sequences structure.
Realize that the technical solution of above-mentioned purpose is as follows.
PORF148 recombinant proteins, successively the amino acid sequence as shown in SEQ ID NO.13-SEQ ID NO.16 pass through Connexon is formed by connecting, and 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 pORF148 recombinant proteins, sequence, including a：Its nucleotide sequence such as SEQ Shown in ID NO.17；b：With the nucleotide sequence coded mutually homotactic protein of a, but due to the degeneracy of genetic code and a The different sequence of nucleotide sequence；C:Substituting, lacking for one or more bases is carried out to nucleotide sequence shown in above-mentioned a or b It loses, the nucleotide sequence of addition modification.
The preparation method of above-mentioned pORF148 recombinant proteins, comprises the following steps：
Expressing gene sequence comprising the above-mentioned coding pORF148 recombinant proteins is inserted by HindIII/XhoI double digestions Enter pET32a (+) carrier, construction recombination plasmid pET32a-mod pORF148；
DH5 α are transferred to again, and PCR screening positive transformants bacterial strains extract plasmid, are transferred to respectively after positive strain sequencing identification BL21 (DE3) expresses bacterial strain；
BL21 (DE3) the expression bacterial strains of picking conversion pET32a-mod pORF148 are inoculated in the LB culture mediums of Amp resistances Middle overnight incubation is inoculated in LB fresh cultures, add IPTG induced expressions, purifying to get.
Encode the sequence such as SEQ ID NO.17 of the expressing gene of the pORF148 recombinant proteins.
It is detected it is a further object of the present invention to provide a kind of in fancy carp serum for the specific antibody of CyHV-3 ORF148 Kit.
Specific technical solution is as follows：
The kit of the serum specific antibody of fancy carp is detected, includes the pORF148 recombinant proteins.
In one of the embodiments, the kit is ELISA kit, including the useful pORF148 restructuring egg White coated ELISA Plate.
In one of the embodiments, the polyclonal antibody of the anti-fancy carp IgM of mouse is further included.
On the basis of the present invention passes through to the numerous studies of CyHV-3, inventor has intercepted CyHV-3 pORF148 whole eggs Epitope advantage section in Bai Xulie has carried out prokaryotic expression.Expression effect is good.It is further shown through ELISA, this section The albumen (its coding DNA after optimizing is SEQ ID NO.17) of short expression can be used as envelope antigen, can examine well The serum specific antibody generated after fancy carp is immunized in the DNA vaccination for surveying CyHV-3 ORF148 complete encoding sequences structure.
Description of the drawings
Fig. 1 pORF148 sequence analyses：Wherein, arrow represents signal peptide cutting site (SignalP4.1 software predictions), Bold Italic marks the B cell epitope advantage section that cross-film sequence (TMHMM software predictions) wave marks DNAstar predictions, cloudy Shadow shows the B cell epitope of ABCpred predictions, and box marks the B cell epitope of BepiPred predictions, and runic marks final prediction B cell epitope advantage section.
Fig. 2 fusion DNA vaccine electrophoretograms：Wherein, M:Marker, 1,2：MORF148 fusion DNA vaccines expand.
The recombination expression of Fig. 3 pORF148：Wherein, M:Protein Marker, 1：Pet32a (+) is not induced, and 2： Pet32a (+) induction, 3：Pet32a (+)-compORF148 is not induced, and 4：Pet32a (+)-compORF148 is induced, and 5： Pet32a (+)-tORF148 is not induced, and 6：Pet32a (+)-tORF148 is induced, and 7：Pet32a (+)-mORF148 is not induced, and 8： Pet32a (+)-mORF148 is induced.
The Western blot identifications of Fig. 4 recombination expressions pORF148：Wherein, M:Protein Marker, 1：pet32a (+)-mORF148 is induced.
Fig. 5 fancy carps serum IgM purifies：Wherein, M:Albumen Marker, 1：Do not purify fancy carp serum, 2：Washing percolation liquid, 3： Eluent.
Specific embodiment
Unless otherwise defined, technical field of all technical and scientific terms used in the present invention with belonging to the present invention The normally understood meaning of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality The purpose of example is applied, is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed The arbitrary and all combination of project.
For the ease of understanding the present invention, the present invention will be described more fully below.But the present invention can be with perhaps Mostly different form is realized, however it is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments is to make To the understanding more thorough and comprehensive of the disclosure.
Reagent or raw material used in following embodiment unless otherwise specified, derive from commercially available.
Embodiment：
First, materials and methods
1.CyHV-3ORF148 complete sequence protokaryon induced expressions
ORF148 genes (GenBank No.KP004903) are analyzed, design primer (table 1).With CyHV-3- HZ419 DNA are template, using Prime Star Max high-fidelity enzymes, are expanded with primer 148Hind3CF/148XholCR (table 1) Increase ORF148 complete encoding sequences.Response procedures use：94 DEG C of 5min pre-degenerations；94 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 90s, 35 Xun Huan；72 DEG C of 5min overall elongations, 4 DEG C of preservations.
1 ORF148 amplimers sequence (5 ' -3 ') of table
Table 1 Sequence of ORF148gene amplification
Above-mentioned amplified production is inserted into pET32a (+) carrier, the recombinant plasmid of structure by HindIII/XhoI double digestions PET32a-compORF148 is named as, is transferred to DH5 α, PCR screening positive transformants bacterial strains extract matter after positive strain sequencing identification Grain is transferred to BL21 (DE3) expression bacterial strains.Picking converts bacterial strain, is inoculated in overnight incubation in the LB culture mediums of Amp resistances, then presses 1:100 ratios are inoculated in 100mLLB fresh cultures, 37 DEG C, and 220rpm/min, which is cultivated to bacterium solution OD values, reaches 0.4-0.5, 0.8mM IPTG are separately added into 28 DEG C of induced expressions.
2.pORF148 the construction and expression of sequence analysis, codon optimization and recombinant plasmid
ORF148 genes and protein sequence (GenBank No.KP004903, AJK93609) are analyzed, predict antigen Epitope advantage section.Finally, according to the experience of inventor, by repeatedly assessing, determining antigen fragment, (SEQ ID NO.13 are extremely SEQ ID NO.16),
Using the tORF148 of the codon transformation and optimization of Jin Weizhi synthesis as template, with primer 148TXho1R/ The tORF148 (SEQ ID NO.17) of 148THind3F (table 2) amplification optimizations.Amplification condition, vector construction, derivational expression method With complete sequence protokaryon derivational expression method, constructed recombinant plasmid is respectively designated as pET32a-tORF148.
2 ORF148t amplimers sequence (5 ' -3 ') of table
Table 2Sequence ofORF148t gene amplification(5’-3’)
3.pORF148B cell epitope advantages section fusion amplification, the construction and expression of recombinant plasmid
3.1.pORF148B cell epitope advantage section fusion amplification
B cell epitope advantage section prediction result design primer in 2, several B cell epitope advantage sections are adopted It is connected with Gly-Gly-Gly-Gly-Ser (G4S) flexible section, with the tORF148 of the codon transformation and optimization of Jin Weizhi synthesis For template, fusion amplification is carried out using fusion DNA vaccine technology, the primer is shown in Table 3, and response procedures are：94 DEG C of 5min pre-degenerations；94 DEG C 30s, 59 DEG C of 30s, 72 DEG C of 90s, 35 cycles；72 DEG C of 2min overall elongations, 4 DEG C of preservations.Obtain B cell epitope fusion section mORF148。
3 ORF148m amplimers sequence (5 ' -3 ') of table
Table 3Sequence of ORF148m gene amplification
3.2.pORF148B the construction and expression of cell epitope advantage section recombinant plasmid
With complete sequence protokaryon derivational expression method, constructed recombinant plasmid is named as vector construction, derivational expression method
pET32a-mORF148.After obtaining positive expression product, albumen is carried out up to system handbook with reference to Novagen pet sheets Expression and soluble analysis.The protein expressioning product under optimum condition of the expression is chosen, it is pure using Novagen His Bind albumen Change kit to be purified, purifying protein -80 DEG C of preservations after BCA kit quantifications.
4. recombinate the Western-blot analyses of pORF148m
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 1:37 DEG C of incubation 2h of anti-His labels mouse monoclonal antibody of 1000 diluted HRP marks, PBST is detected after washing film 5 times with Bio-Rad bioluminescent reagents box.
5. fancy carp serum specific antibody detects the foundation of ELISA method
5.1. the purifying of fancy carp serum IgM
Fancy carp serum is gathered, with 4moLL-1NaCl (pH8.3) is mixed in equal volume, and 1mL is taken to add in 2moLL-1NaCl (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-1NaCl (pH8.3) washs 10 column beds, 0.05moLL-1Gly is eluted, collection eluent, and 1:50 add in Tris- HCl (pH8.0) adjusts pH.Purified product carries out SDS-PAGE electrophoretic analysis.Purify band Qie Jiao Hou Songmeiji biotech firms into Row LC-MS/MS is analyzed.
5.2. the preparation of the polyclonal antibody of the anti-fancy carp IgM of mouse
Using the carp IgM of above-mentioned purifying as mice immunized with antigen (4), every is immunized 3 times altogether, every minor tick 2 weeks, the 1st time Antigen adds isometric Freund's complete adjuvant, latter 2 times plus isometric incomplete Freund's adjuvant, and immunizing dose is 50 μ g-1, Immunization route is back and subcutaneous abdomen multi-point injection.3 days booster immunizations before blood sampling are injected intraperitoneally, agent with adjuvant antigen is not added with It measures as 50 μ g only-1.Eyeball blood sampling is extractd, (Beijing Webster wins the limited public affairs of intelligent chromatography science and technology to separation serum using rProtein A Department) purified mouse IgG, -80 DEG C of preservations.
6.ORF148DNA vaccines are immunized
With reference to the method for Liu Zhenxing (2015), Bgl II, III digestions of Hind are utilized after ORF148 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-ORF148 of structure The fancy carp of weight 20g, immunizing dose are 3 μ g/ tails, and serum, -80 DEG C of preservations are gathered after 3 weeks immune.
7. indirect EILSA methods detect immune serum antibody titer
The pORF148m (pET32a-mORF148) of purifying is diluted to 320 μ g/mL with CBS, 100 μ L/ holes coated elisa plates, 4 DEG C overnight, and PBST is washed 3 times；Confining liquid (1%BSA) is added in, 37 DEG C are closed 1.5h, and PBST is washed 3 times；Add in 1:300 dilutions 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 carps of mouse are more Clonal antibody (4.2 prepare), 37 DEG C are incubated 2.5h, and PBST is washed 5 times；Add in 100 μ L/ holes 1:8000 dilution HRP mark goat-antis Mouse IgG, 37 DEG C are incubated 2h, and PBST is washed 5 times；Add in 100 μ L/ holes TMB working solutions, 37 DEG C of incubation 30min.Add in 50 μ L/ Hole terminate liquid detects OD450.
2nd, interpretation of result
1.CyHV-3 ORF148 complete sequence protokaryon induced expressions
PAGE electrophoresis results show in the selected each temperature of this experiment, IPTG concentration and induction time, all do not have Have and obtain CyHV-3ORF148 expression products.
2.pORF148 the construction and expression of sequence analysis, codon optimization and recombinant plasmid
HZ419 plants of ORF148 complete encoding sequence overall length 1803bp of CyHV-3, encoding proteins include 601 amino acid.It should Gene includes 1 signal peptide cutting site and 1 transmembrane segment (Ile561-Leu591).Rare codon analysis shows that, Low abundance codon (threshold=10) has 134 in CyHV-3 ORF148 complete encoding sequences, to obtain expression product, These low abundance codons are all replaced with Escherichia coli preference codon by us, and by signal peptide, transmembrane region and cross-film Sequence after the adjacent hydrophobic region in area removes send progress vector construction and induced expression after genome company's synthesis segment.As a result show Show, after carrying out codon transformation and removing signal peptide, transmembrane region and hydrophobic structure, still cannot obtain expression product.
3.pORF148 sequence analysis is predicted with B cell epitope advantage section
3.1.pORF148B cell epitope advantage section fusion amplification
HZ419 plants of ORF148 complete encoding sequence overall length 1803bp of CyHV-3, encoding proteins include 601 amino acid, most 4 B cell epitope advantage sections of definite pORF148 eventually：Asp157-Vel205, Cys220-Thr241, Vel262- Glu353, Vel412-Ser544 (Fig. 1).After fused PCR amplification, we obtain 4 B cell epitope advantage sections to connect Segment ORF148m (Fig. 2).
DIGASAYTWYRNGVFADTTSTNEYITVVAGRYTCEPTGSTGTLSDPVWV SEQ ID NO.13
GGGGS
CPNGAITRIHEATSKTYTDIHT SEQ ID NO.14
GGGGS
VSTRGNCPVKLSACNNNEQLTCANQAPIPVLNTCNRVYSYFCPDHYYFSYTAVNSTGASLGLTPVDANEPRRIEMPG TNGTVYRIYCGNDFE SEQ ID NO.15
GGGGS
VKRSAPANPLLVSTGTSELIKVAELSDFTDWECAVFTNPGDSQGIRTRLFNVTQLTTTTATTLTTPSTPPTTPTVIT PPTNQSITPTPPTTPTTTPTTPNITTPTTPSTPSTTTPTTPSTPTSTSTSTSTSTS SEQ ID NO.16
The codon optimised sequence of pORF148B cell epitope advantage sections
GATATCGGCGCCAGCGCATATACCTGGTACCGCAATGGCGTGTTCGCCGACACCACCAGCACAAACGAGTATATCAC CGTGGTGGCCGGTCGCTATACCTGCGAGCCGACAGGCAGCACCGGTACACTGAGTGATCCTGTGTGGGTTGGCGGCG GCGGCAGCTGCCCGAACGGTGCAATCACCCGTATTCACGAAGCCACCAGCAAAACCTACACCGACATCCATACCGGC GGCGGCGGCAGCGTGAGTACCCGTGGTAACTGCCCGGTGAAACTGAGCGCCTGCAACAACAACGAACAGCTGACCTG CGCCAATCAGGCCCCGATTCCGGTGCTGAATACCTGCAACCGTGTGTACAGCTATTTCTGCCCGGATCACTATTATT TTAGCTACACCGCAGTTAATAGCACCGGCGCCAGCCTGGGTCTGACCCCGGTTGATGCAAATGAGCCGCGTCGCATT GAGATGCCGGGCACCAACGGCACCGTGTATCGTATCTACTGCGGCAACGATTTTGAGGGCGGCGGCGGCAGCGTGAA ACGTAGCGCACCGGCCAATCCGCTGCTGGTTAGCACAGGCACCAGCGAACTGATCAAAGTGGCCGAACTGAGTGACT TCACCGATTGGGAATGCGCAGTGTTCACAAACCCGGGCGATAGCCAGGGCATTCGTACCCGCCTGTTCAACGTGACC CAGCTGACCACAACCACAGCCACCACCCTGACCACACCTAGTACCCCGCCGACAACCCCGACCGTGATTACCCCGCC GACCAACCAAAGTATCACCCCGACACCGCCGACCACACCTACCACCACCCCGACCACACCGAATATCACAACACCGA CCACCCCGAGTACCCCGAGTACCACCACCCCGACCACACCGAGCACCCCGACCAGTACCAGTACCAGTACCAGCACC AGCACCAGC SEQ ID NO.17。
3.2.pORF148B the expression of cell epitope advantage section
Pet32a (+)-compORF148, pet32a (+)-tORF148 merge egg with what pet32a (+)-mORF148 was expressed White theoretical molecular weight is respectively 83.5kda, 75.6kDa, and 52.1kDa, using 0.8mM IPTG, 28 DEG C induce 4h only pet32a (+)-modORF148 visible apparent band of expression (Fig. 3), soluble analysis near purpose band show albumen with inclusion body Form is expressed.Using Novagen His Bind protein purification kits destination proteins, the murine monoclonal through anti-His labels Antibody Western-blot is analyzed, and apparent band (Fig. 4) is hybridized at 52.1kD.
3. recombinate applications of the pORF148 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. 5), 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 the present invention as detection antibody, using the restructuring pORF148 of purifying as bag By antigen, conventionally, indirect EILSA methods detection fancy carp immune serum antibody titer is established, the results showed that 5 tails are immunized Fancy carp serum OD450 is is respectively 0.415,0.439,0.466,0.423,0.419, non-immune serum (negative control) OD450 For 0..175, P/N ＞ 2.1.More than in triplicate, testing result is stablized, and this method can evaluate fancy carp 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, the scope that this specification is recorded all is considered to be.
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 come for those of ordinary skill in the art 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 Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
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Thr Thr Pro Asn Ile Thr Thr Pro Thr Thr Pro Ser Thr Pro Ser Thr
100 105 110
Thr Thr Pro Thr Thr Pro Ser Thr Pro Thr Ser Thr Ser Thr Ser Thr
115 120 125
Ser Thr Ser Thr Ser
130
<210> 17
<211> 933
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
gatatcggcg ccagcgcata tacctggtac cgcaatggcg tgttcgccga caccaccagc 60
acaaacgagt atatcaccgt ggtggccggt cgctatacct gcgagccgac aggcagcacc 120
ggtacactga gtgatcctgt gtgggttggc ggcggcggca gctgcccgaa cggtgcaatc 180
acccgtattc acgaagccac cagcaaaacc tacaccgaca tccataccgg cggcggcggc 240
agcgtgagta cccgtggtaa ctgcccggtg aaactgagcg cctgcaacaa caacgaacag 300
ctgacctgcg ccaatcaggc cccgattccg gtgctgaata cctgcaaccg tgtgtacagc 360
tatttctgcc cggatcacta ttattttagc tacaccgcag ttaatagcac cggcgccagc 420
ctgggtctga ccccggttga tgcaaatgag ccgcgtcgca ttgagatgcc gggcaccaac 480
ggcaccgtgt atcgtatcta ctgcggcaac gattttgagg gcggcggcgg cagcgtgaaa 540
cgtagcgcac cggccaatcc gctgctggtt agcacaggca ccagcgaact gatcaaagtg 600
gccgaactga gtgacttcac cgattgggaa tgcgcagtgt tcacaaaccc gggcgatagc 660
cagggcattc gtacccgcct gttcaacgtg acccagctga ccacaaccac agccaccacc 720
ctgaccacac ctagtacccc gccgacaacc ccgaccgtga ttaccccgcc gaccaaccaa 780
agtatcaccc cgacaccgcc gaccacacct accaccaccc cgaccacacc gaatatcaca 840
acaccgacca ccccgagtac cccgagtacc accaccccga ccacaccgag caccccgacc 900
agtaccagta ccagtaccag caccagcacc agc 933

Claims (10)

1.pORF148 recombinant proteins, it is characterized in that, the amino acid sequence as shown in SEQ ID NO.13-SEQ ID NO.16 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 pORF148 recombinant proteins described in claim 1, it is characterized in that, sequence, including a： Its nucleotide sequence is as shown in SEQ ID NO.17；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 substitution of multiple bases, missing, the nucleotide sequence of addition modification.
4. the preparation method of pORF148 recombinant proteins described in claim 1, it is characterized in that, comprise the following steps：
Expressing gene sequence comprising the coding pORF148 recombinant proteins described in claim 3 is double by HindIII/XhoI PET32a (+) carrier, construction recombination plasmid pET32a-mod pORF148 are inserted into digestion；
DH5 α are transferred to again, and PCR screening positive transformants bacterial strains extract plasmid, are transferred to BL21 respectively after positive strain sequencing identification (DE3) bacterial strain is expressed；
BL21 (DE3) the expression bacterial strains of picking conversion pET32a-mod pORF148, are inoculated in the LB culture mediums of Amp resistances and train Support 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 pORF148 recombinant proteins The sequence of gene is as shown in SEQ ID NO.17.
6. preparation method according to claim 5, it is characterized in that, as described in the coding shown in SEQ ID NO.22 The expressing gene of pORF148 recombinant proteins its expand to obtain by SEQ ID NO.3 and SEQ ID NO.4.
7. claim 1-2 any one of them pORF148 recombinant proteins are preparing the serum specific antibody of detection fancy carp Application in kit.
8. the kit of the serum specific antibody of fancy carp is detected, it is characterized in that, include described in claim 1 or 2 PORF148 recombinant proteins.
9. the kit of the serum specific antibody of detection fancy carp according to claim 8, it is characterized in that, the kit For ELISA kit, including the useful claim 1 or 2 coated ELISA Plate of pORF148 recombinant proteins.
10. the kit of the serum specific antibody of detection fancy carp according to claim 8 or claim 9, 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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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

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