CN108530522A

CN108530522A - A kind of OmpK multi-epitopes polypeptide, construction method and its application of recombination

Info

Publication number: CN108530522A
Application number: CN201810208626.XA
Authority: CN
Inventors: 张玉晴; 许如苏; 汪天杰; 张峥嵘; 吴松浩; 许晓升; 沈烨; 周铮宇
Original assignee: SHANTOU ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU PEOPLE'S REPUBLIC OF CHINA
Current assignee: Shantou customs of the people's Republic of China
Priority date: 2018-03-14
Filing date: 2018-03-14
Publication date: 2018-09-14
Anticipated expiration: 2038-03-14
Also published as: CN108530522B

Abstract

The present invention relates to biomedicine fields, specifically, the present invention relates to recombinant polypeptide, construction method and its applications, more specifically, the present invention provides the nucleotide of a kind of OmpK multi-epitopes polypeptide of recombination and its coding, carrier, cell, vaccine and its purposes in the product for preventing vibrio infection.Experiments have shown that; OmpK multi-epitope polypeptides of recombination provided by the invention or derivatives thereof and vaccine; have the characteristics that safe and efficient; it has vibrio parahemolyticus very high immunogenicity; animal through this multi-epitope polypeptide immune; antibody in vivo containing a large amount of specificity, reaches 90% or more to immune animal Effective Vate of Protection；Moreover, the OmpK multi-epitope polypeptides of the recombination improve the resistivity for receiving the animal of this multi-epitope polypeptide immune to vibrio parahemolyticus, there is good immunoprotective effec, this has great importance in terms of prevention and control aquatic livestock infectious disease.

Description

A kind of OmpK multi-epitopes polypeptide, construction method and its application of recombination

Technical field

The present invention relates to biomedicine fields, specifically, the present invention relates to recombinant polypeptide, construction method and its application, More particularly it relates to which the nucleotide of recombinant polypeptide and its coding, carrier, cell, vaccine are in the production for preventing vibrio infection Purposes in product.

Background technology

Vibrio parahemolyticus (Vibrio parahaemolyticus, VP) is a kind of gram-negative of vibrionaceae vibrio bacterial Property bacterium, is commonly called as Halophiles, vibrio parahemolyticus is widely present in marine environment and rivers mouth, can infect fish, shrimp and shellfish The multiple aquatic animals such as class cause the diseases such as red body diseases of shrimps, fish skin ulcer, are the main diseases for threatening sea-farming industry One of opportunistic pathogen has brought tremendous economic losses to culture fishery.The bacterium infects as many as 48 kinds of seawater fish, and incidence is high, Epidemic Scope is wide, and harm is serious, be limit the marine economic animals aquaculture developments such as China marine fishes shellfish it is main because One of element.Vibrio parahemolyticus or a kind of important food-borne pathogens are most common in the food poisoning of China's Coastal Areas A kind of pathogen, prodigious threat is constituted to human health.Network data is monitored according to China's food origin disease, in portion of China Divide in coastal area microorganism food poisoning example, the food poisoning case that vibrio parahemolyticus causes holds pride of place, and is permitted The main inducing for the disease that more Asian countries's marine products cause.

For a long time, the main means of control aquatic livestock bacterial disease are to use antibacterials, but with antibacterials Reusability, bacterium generates drug resistance, and antibacterial curative effect unobvious lead to the large quantities of death of infected animal.

Vaccine inoculation is to prevent one of the important method of fish infectious disease, and the research of domestic vaccines for fish is started late, greatly The research and development of most vaccines mainly also rest on laboratory research stage, and mostly full bacterium inactivated vaccine.Although inactivated vaccine is very Safety, but there are immune effects poor, the larger disadvantage of side effect.

Therefore, the vaccine for preventing fish infectious disease of structure efficiently, safe has prevention fish infectious disease important Meaning.

Invention content

In vibrio parahemolyticus there are a kind of relatively conservative important outer membrane protein (outer membrane protein, Omp) K, the albumen are a kind of distinctive outer membrane proteins of vibrio type, and nineteen ninety-five, Inoue T are for the first time in vibrio parahemolyticus Middle discovery is simultaneously named, which is widely present in the vibrio parahemolyticus of different serotypes, and is deposited in Vibrio There is immune cross-reactivity in vibrios, and this immunological cross-reaction has species specificity.OmpK albumen is not only It is a kind of outer membrane protein shared in vibrios, and OmpK genes are highly conserved in vibrios.Yang Zhihui in 2006 etc. is from gene It confirms that OmpK is widely present in seawater fish kinds of pathogenic vibrio in level, is a kind of preferable common antigen of immunogenicity. 2007, the vibrio parahemolyticus ompK of Mao Zhijuan recombinations was immunized Larimichthys crocea and obtains ideal immune protective effect, discloses OmpK is the important protective antigens of vibrio parahemolyticus in natural immunity.The technical problem to be solved in the present invention is to provide OmpK multi-epitope polypeptides of a kind of recombination or derivatives thereof, the OmpK nucleotide of recombination, OmpK nucleotide containing recombination exist The method of the OmpK multi-epitope polypeptides that carrier and cell, kit and its application and structure recombinate or derivatives thereof.

First aspect present invention provides a kind of OmpK multi-epitope polypeptides of recombination or derivatives thereof, the polypeptide or its spread out Biology is selected from：

1) include SEQ ID NO：8-SEQ ID NO：Amino acid fragment sequence shown in 13；

2) the amino acid fragment sequence for including has at least one or several amino compared with amino acid fragment in (1) respectively The substitution of acid lacks and ors add, and the polypeptide has the immunogenicity to vibrios；

3) the amino acid fragment sequence for including respectively from vibrio parahemolyticus, and respectively with SEQ ID NO：8-SEQ ID NO：13 at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homogeneity；

4) derivative of polypeptide, is the derivative of (1)-(3) any polypeptide, and the polypeptide derivative has pair The immunogenicity of vibrios.

The present invention relates to the derivatives of polypeptide.For example, the polypeptide of the present invention can contain modification, such as glycosylation, side chain Oxidation or phosphorylation modification；As long as the modification does not influence the biological activity of the peptide, that is, there is the identification of B cell epitope Active (immunogenicity).The example of the derivative of the polypeptide further includes carrying radioactive label, biotin labeling or fluorescence mark The polypeptide of note.In addition, present invention also contemplates that the hydrate of the polypeptide, solvate or physiologically acceptable salt.

In a preferred example, described polypeptide or derivatives thereof also includes joint sequence 1, and the joint sequence is located at amino Between acid fragment sequence.

In a preferred example, the joint sequence 1 is SEQ ID NO：Sequence shown in 14.

In a preferred example, the amino acid fragment sequence is according to SEQ ID NO：8-SEQ ID NO：13 sequences are successively Connection.

In a preferred example, the polypeptide is SEQ ID NO：Sequence shown in 15.

Second aspect of the present invention provides a kind of OmpK nucleotide of recombination, and the nucleotide includes that coding is described above Polypeptide or derivatives thereof.

In a preferred example, coding SEQ ID NO：8—SEQ ID NO：The nucleosides of amino acid fragment sequence shown in 13 Acid fragment is respectively SEQ ID NO：1-SEQ ID NO：Nucleotide sequence shown in 6 also includes joint sequence 2, the connector Sequence 2 is between nucleotide sequence；.

In a preferred example, the joint sequence 2 is SEQ ID NO：It is nucleotide sequence coded shown in 7.

In a preferred example, the nucleotide fragments are according to SEQ ID NO：1-SEQ ID NO：7-SEQ ID NO： 2-SEQ ID NO：7-SEQ ID NO：3-SEQ ID NO：7-SEQ ID NO：4-SEQ ID NO：7-SEQ ID NO：5-SEQ ID NO：7-SEQ ID NO：It is sequentially connected with shown in 6.

In a preferred example, the nucleotide 5 ' end further includes initiator sequences.

In a preferred example, the initiator sequences are ATG.

In a preferred example, 5 ' ends of the initiator sequences further include cleavage sequence.

In a preferred example, the cleavage sequence at 5 ' ends of the initiator sequences is I restriction endonuclease recognition sequences of BamH.

In a preferred example, the 3 ' ends of the termination codon subsequence further include cleavage sequence.

In a preferred example, the cleavage sequence at the 3 ' ends of the termination codon subsequence is that III digestions of Hind identify sequence Row.

In a preferred example, the nucleotide also includes protected nucleoside acid sequence.

In a preferred example, the protected nucleoside acid sequence is CGC and GGG.

In a preferred example, the sequence of the nucleotide is SEQ ID NO：Sequence shown in 16.

Third aspect present invention provides a kind of carrier, and the carrier includes nucleotide sequence recited above.

In a preferred example, the carrier is pET-32a-SEQ ID NO：16, i.e. pET-32a-repis.

Fourth aspect present invention provides a kind of cell, which is characterized in that the cell contains the load described in claim 5 Body.

In a preferred example, the cell is pET-32a-SEQ ID NO：16/E.coli Rosetta cells, as PET-32a-repis/E.coli Rosetta (DE3) cell, i.e. E.coli Rosetta (DE3) contain pET-32a-repis The cell of plasmid.

Fifth aspect present invention provides a kind of vaccine, the vaccine include preceding claim epitope polypeptide or its spread out Biology.

In a preferred example, the vaccine also includes adjuvant.

It is appreciated that the used adjuvant of the present invention plays the role of increasing multi-epitope immunogenicity of polypeptides, as long as rising The substance acted on to this all can serve as adjuvant and use, the including but not limited to aluminium hydroxide of suitable adjuvant, not formula adjuvant, CpG, cholera toxin, salmonella toxin etc., but not limited to this.

Sixth aspect present invention provides a kind of kit, and the kit includes polypeptide recited above or its derivative Object, carrier recited above, cell recited above, and/or, the vaccine described above stated.

Seventh aspect present invention provides polypeptide recited above or derivatives thereof, nucleotide recited above, institute above Carrier, the cell recited above stated, and/or, the application in the product of vaccine prevention vibrio infection recited above.

In a preferred example, the vibrios is vibrio parahemolyticus.

Eighth aspect present invention provides a kind of method of the OmpK multi-epitope polypeptides or derivatives thereof of structure recombination, described Method comprise the following steps：

(1) the sequential structure feature for utilizing software analysis OmpK albumen, selects the linear epitope of the B cell of OmpK albumen；

(2) according to linear epitope, corresponding nucleotide fragments are designed and its are put in order, and design segment jointing, Restriction enzyme site, protected nueleotide, initiation codon, the position relationship of terminator codon and particular sequence；

(3) nucleotide sequence designed in (2) is obtained；

(4) polypeptide of (3) nucleotide sequence coding is obtained；

In a preferred example, the polypeptide in the step (4) is obtained by following steps：A. core step (3) obtained Thuja acid is connect with expression vector；B. conversion carrier obtains the polypeptide of expression.

In a preferred example, the nucleotides sequence of the step (3) is classified as nucleotide sequence recited above.

In a preferred example, the nucleotides sequence of the step (3) is classified as SEQ ID NO:Sequence shown in 16.

In a preferred example, the polypeptide sequence of the step (4) is polypeptide recited above.

In a preferred example, the polypeptide sequence of the step (4) is SEQ ID NO:Sequence shown in 15.

In a preferred example, the expression vector is pET-32a (+).

In a preferred example, the software is DNAstar protean softwares, is used for the two level of comprehensive analysis OmpK albumen Structure, flexibility, surface possibility, hydrophily and antigenic index.

In the present invention, " expression vector " refers to empty carrier；" carrier ", " recombinant expression plasmid ", " recombinant expression carrier " " recombinant vector " refers to the carrier containing recombination sequence.

The invention has the advantages that:

The present invention provides OmpK multi-epitope polypeptides of a kind of recombination or derivatives thereof and vaccines, the experiment proved that, tool Have the characteristics that safe and efficient, it has very high immunogenicity to vibrio parahemolyticus, the animal through this multi-epitope polypeptide immune, Antibody in vivo containing a large amount of specificity, reaches 90% or more to immune animal Effective Vate of Protection.The multi-epitope polypeptide improves Receive resistivity of the animal to vibrio parahemolyticus of this multi-epitope polypeptide immune, there is good immunoprotective effec. This has great importance in terms of prevention and control aquatic livestock infectious disease.

The present invention also provides a kind of methods of OmpK multi-epitope polypeptides of structure recombination or derivatives thereof, pass through this side Method can filter out a kind of multi-epitope polypeptide fragment of albumen, be connected by suitable connector, and structure has and safely and effectively has There is the multi-epitope polypeptide of immunogenicity.

Kit provided by the invention can be effectively used for prevention and control vibrio parahemolyticus infection, reduce because secondary molten The death rate caused by courageous and upright vibrio infection.

Description of the drawings

Fig. 1：The nucleotide homology of the OmpK albumen of 13 plants of vibrio parahemolyticus is analyzed.

Fig. 2：The amino acid identity of the OmpK albumen of 13 plants of vibrio parahemolyticus is analyzed.

Fig. 3：The secondary structure prediction figure A of OmpK albumen:Alpha helical region domain；B:β-pleated sheet region；T:Corner area；C:Randomly Then curled regions；F:Flexible region.

Fig. 4：The hydrophily of OmpK albumen, the prognostic chart of antigenicity and surface possibility, Hydrophilicity：It is hydrophilic Property, Surface Probability:Surface possibility and antigenic index：Antigenic index.

Fig. 5：The OmpK protein B cell epitopes of prediction, Hydrophilicity：Hydrophily, Surface Probability:Surface possibility and antigenic index：Antigenic index, A:Alpha helical region domain；B:β-pleated sheet region；T: Corner area；C:Random coil region.

Fig. 6：The PCR qualification figures of recombinant expression plasmid pET-32a-repis, M.DNA marker；1. recombinant expression plasmid The pcr amplification product of pET-32a-repis；2. the pcr amplification product of empty plasmid pET-32a.

Fig. 7：The PCR qualification result figures of recombinant expression plasmid pET-32a-repis, M.DNA marker；1. empty plasmid The pcr amplification product of pET-32a；2. the pcr amplification product of recombinant expression plasmid pET-32a-OmpK.

Fig. 8：Recombinate the expression SDS-PAGE testing results of OmpK albumen, rEPIS albumen and label protein, M. protein marks Quasi-molecule amount；1.pET-32a-OmpK induced precipitations；2.pET-32a-OmpK induces supernatant；3.pET-32a-OmpK is not induced； 4.pET-32a is not induced；5.pET-32a induces supernatant；6.pET-32a induced precipitations；7.pET-32a-repis is not induced； 8.pET-32a-repis induces supernatant；9.pET-32a-repis induced precipitations.

Fig. 9：Recombinate the SDS-PAGE figures after purification of OmpK albumen, rEPIS albumen and label protein, M. protein standards Molecular weight；The His-Tag albumen of 1,2. purifying；The recombination REPIS albumen of 3,4. purifying；The recombination OmpK albumen of 5,6. purifying.

Figure 10：Recombinate rEPIS albumen Western blot analysis result figures, M. protein standard markers；1. purifying Recombinate rEPIS albumen.

Specific implementation mode

Unless specifically indicated, term used herein has the general sense in fields of the present invention.

Below with reference to specific embodiments and the drawings, the present invention will be described, it should be noted that these embodiments are only It is illustrative, and is not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to Conventional laboratory conditions, or the condition according to manufacturer's specification suggestion.Reagents or instruments used without specified manufacturer, Being can be with conventional products that are commercially available.

The key instrument equipment and reagent used in embodiment

Nucleic acid restriction endonuclease (BamH I, Hind III), ExTaq polymerases and T4 ligases are purchased from Bao doctor's object skill Art (Beijing) Co., Ltd；Bacterial genomes DNA extraction kit, DNA gel QIAquick Gel Extraction Kit and plasmid purification kit purchase From TIANGEN Biotech (Beijing) Co., Ltd.；Non- pre-dyed standard protein and pre-dyed standard protein are that Beijing health is century biology Science and Technology Ltd.'s product；Ni-NTA purification medias are purchased from Nanjing Genscript Biotechnology Co., Ltd.；Ammonium persulfate, TEMED, beta -mercaptoethanol etc. are purchased from BBI companies of the U.S.；MarkerDL2000, ampicillin (Amp), Coomassie brilliant blue R250, lauryl sodium sulfate (SDS), 40% acrylamide solution, trishydroxymethylaminomethane (Tris alkali), glycine, miaow Azoles, dithiothreitol (DTT) (DTT), ethylenediamine tetra-acetic acid (EDTA) and isopropylthiogalactoside (IPTG) are purchased from Shanghai life Object Engineering Co., Ltd.

CLASS II A/B3 type Biohazard Safety Equipments；Bole's MyCycler PCR instruments；Full automatic gel imaging analysis instrument； III type Horizontal electrophoresis tank (Liuyi Instruments Plant, Beijing) of DYY-8C types electrophoresis apparatus and DYY-；High-speed refrigerated centrifuge；DNP-9082 electricity Hot constant incubator；II ultrasonic cell-break machines of JY92- (Ningbo Xin Zhike devices research institute)；DK-8D electric heating constant temperature sinks； Vertical pressure steam sterilizer.

Embodiment 1 prepares nucleotide sequence

The design and synthesis of 1 OmpK protein epitope tandem genes

(1) selection of OmpK protein amino acid sequences

Log in the nucleotide sequence of search vibrio parahemolyticus OmpK genes in US National Bioinformatics Institute (NCBI) And amino acid sequence, through searching, the complete nucleotide sequence for the vibrio parahemolyticus OmpK that NCBI is logged in shares 13, such as table 1 Shown, open reading frame (ORF) length encodes 264~274 amino acid, OmpK molecular weight of albumen between 792~822bp Between 29.4KD~30.5KD, the gene order accession number of the OmpK albumen of ATCC17802 is HM044386.1, and ORF length is 819bp, encodes 273 amino acid, and OmpK molecular weight of albumen is 30.3KDa.Utilize 13 kinds of DNAStar Megalign softwares pair The nucleotide sequence and amino acid sequence of OmpK genes carry out homologous comparisons and analyze, and analysis result is as depicted in figs. 1 and 2, from than To result as can be seen that the nucleotide and amino acid identity of the OmpK albumen of 13 plants of vibrio parahemolyticus are respectively 72.1% ~100% and 75.8%~100%, ATCC17802 and another 12 plants of nucleotide and amino acid identity are higher, respectively 76.1%~99.4% and 79.3%~99.6%.Vibrio parahemolyticus reference culture ATCC17802 is chosen based on comparison result OmpK protein amino acid sequences (HM044386.1) predict its B cell epitope.

Table 1：The complete nucleotide sequence for 13 plants of vibrio parahemolyticus OmpK that NCBI is logged in

Serial number	Accession number	ORF overall lengths (bp)	Bacterium source
				1	CP006008	819	The U.S.
2	CP009847	819	South Korea
				3	CP011406	819	South Korea
4	D61392	792	Japan
				5	DQ016304	819	Zhejiang
6	FJ172213	819	Guangdong
				7	JQ429754	798	India
8	FJ462706	798	Jilin
				9	FJ462709	822	Jilin
10	GU318337	819	Guangdong
				11	GU318338	819	Guangdong
12	HM044386	819	Guangdong
				13	HQ157203	819	Shenzhen

(2) OmpK protein structural analysis

The OmpK albumen of vibrio parahemolyticus reference culture ATCC17802 is analyzed using DNAStar Protean softwares Secondary structure and flexible region, the method that secondary structure analysis uses is Chou-Fasman methods and Garnier-Robson methods, The common region of two methods analysis result, flexible region (Flexibility) is taken to analyze using Karplus-Schulz Method.Analysis result is shown in Fig. 3, A:Alpha helical region domain；B:β-pleated sheet region；T:Corner area；C:Random coil region；F:It is flexible Region.Specific secondary structure location information is shown in Table 2.By table 2 and Fig. 3 as it can be seen that the secondary structure of OmpK albumen include β-pleated sheet, β-bend and random coil structure are a kind of mixed type albumen.It is easily formed the corner area of epitope and random volume Bent region is predominantly located at 3-6,10-12,20-30,33-36,43-46,61-70,77-79,110-113,141-144,154-- 243 amino acid sections of 158,165-169,182-187,193-197,205-207,227-228,231-237,242-；OmpK contains There are multiple flexible regions, is predominantly located at 5-13,25-34,42-46,61-72,86-91,94-97,108-123,131,139- 147,154,164-167,182-197,219-220,226-229,236-242 amino acid sections；β-bend, random coil and Flexible region is often the region to form epitope.

Table 2：The secondary structure of OmpK albumen and the analysis result of flexible region

(3) the B cell Antigen Epitope Prediction of OmpK albumen

Using the OmpK albumen of DNAStar Protean software prediction vibrio parahemolyticus reference cultures ATCC17802 Amino acid pro aqueous (Hydrophilicity), surface possibility (Surface probability) and antigenic index (Antigen-icity index AI), is respectively adopted Kyte-Doolittle methods, Emini methods and Janeson-Wolf methods, table Position prediction result is shown in Fig. 4.The prediction result of three kinds of parameters of OmpK albumen is：It may it was found that meeting hydrophilic parameter >=0, surface Property parameter >=1 and the region of average antigenic index >=0 share 9, be located at 1-4,7-14,24-37,63-69,140- 147,182-188,218-221,225-228,234-239 amino acid section further exclude secondary structure and are located at β-pleated sheet area It is not easy to form the section of epitope in domain, person at β-bend and random coil will be located at and be determined as B cell epitope, obtain OmpK eggs White 6 B cell epitopes, position and its mean antigen index are between 1.98~2.81.The prediction knot of three kinds of parameters of comprehensive analysis Fruit obtains the advantage B cell linear epitope of OmpK albumen in conjunction with the second structure characteristic and flexible region of OmpK albumen, prediction As a result see Fig. 5, the more specific location information of the correlated series of the hydrophily of OmpK albumen, surface possibility and antigenic index is shown in Table 3, the B cell epitope of the OmpK albumen of prediction is shown in Table 4, OmpK protein Bs cell epitope and original sequence (the 13 plants of secondary haemolysis of prediction The amino acid sequence of the OmpK albumen of property vibrios) comparing result is shown in Table 5.

Table 3：The hydrophily of OmpK albumen, the analysis result of surface possibility and antigenic index

Table 4：The OmpK protein B cell epitopes of prediction

Table 5：The OmpK protein Bs cell epitope of prediction and former alignment

(4) the multi-epitope tandem polypeptide design of OmpK albumen

By it is predicted that obtain vibrio mimicus vibrio parahemolyticus reference culture ATCC17802 OmpK albumen epitope sequence Row GGGS (SEQ ID NO：14) (Gly-Gly-Gly-serine) connector connects, by the more of optimization design Epitope series connection peptide amino acid sequence DIHKNDYGGGSNEKGYAESSHDYGGGSPGSDKAGGGGS YDGNKKDWGGGSDDDKGNKGGGSGHKPES(SEQ ID NO：15) nucleotide sequence, SEQ ID NO are converted into：8-SEQ ID NO：14 corresponding nucleotide sequences are respectively GATATCCACAAAAACGATTAC (SEQ ID NO：1), AACGAGAAAGGTTATGCTGAATCTTCTCATGATTAC(SEQ ID NO：2), CCAGGCAGCGACAAAGCTGGC (SEQ ID NO：3), TACGATGGCAACAAGAAAGATTGG (SEQ ID NO：4), GATGACGACAAAGGTAACAAG (SEQ ID NO：5), GGTCACAAACCAGAATCT (SEQ ID NO：6), and SEQ ID NO：15 amino acid sequence is converted into SEQ ID NO：1-SEQ ID NO：The nucleotide sequence of 7 sequences composition, and I restriction enzyme sites of BamH, protectiveness base (CGC) and initiation codon (ATG) are added in gene order 5', is added at the ends 3' III restriction enzyme sites of Hind, protectiveness base (GGG), and after codon optimization, by the nucleotide sequence (expression of final design The nucleotide sequence of rEPIS albumen) SEQ ID NO：16 send to the synthesis of general biosystem (Anhui) Co., Ltd.

It is (single offline：Protectiveness base；It is double offline：BamH I and Hind III；Dotted line：It is initiation codon；Wave：Series connection connector (GGGS))。

2 prepare the nucleotide sequence of OmpK genes

According to NCBI log in vibrio parahemolyticus ATCC17802 bacterial strains OmpK nucleotide sequences (HM044386.1), Using SignaIP4.1 software prediction OmpK protein signal peptide sequences, signal peptide is removed, with the Main Antigenic Region base of OmpK albumen The both sides sequence of cause separately designs upstream and downstream primer, and upstream and downstream primer adds BamHI and HindIII restriction enzyme sites and guarantor respectively Shield property base, sense primer F1 are 5'-CGGGATCCGCAGATTACTCTGACGGCGATAT-3'(SEQ ID NO：17) under, Trip primers F 2 is 5'-CCCAAGCTTTTAGAATCCGTAAGTTACTGCGA-3'(SEQ ID NO：18), scribing line portion in sequence It is divided into restriction enzyme site.According to bacterial genomes DNA extraction kit (article No.：DP302-02；Producer：Tiangeng biochemical technology (north Capital) Co., Ltd) specification operation, extraction ATCC17802 bacterial strains are (in Shantou Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technology The heart) genome is as template, PCR amplification OmpK genes, according to the reaction system of the μ L of system configurations total volume 50.0 shown in table 6：

Table 6：PCR reaction systems

Reagent	Volume
		10×PCR buffer	5.0μL
dNTP	4.0μL
		F1/F2	Each 1 μ L (1 μm of ol/L of final concentration)
ExTaq enzymes	0.2 μ L (final concentration 0.02U/ μ L)
		ddH₂O	37.8μL
Genome	1.0 μ L (2.5 μ g/ μ L of final concentration)

PCR reaction conditions are：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 1min, 52 DEG C of annealing 45s, 72 DEG C of renaturation 45s, altogether 30 cycles；Extend 10min after 72 DEG C.Product carries out recovery purifying acquisition after agarose gel electrophoresis and sequencing identification The PCR product of OmpK genes.

Embodiment 2 prepares expression plasmid

1. endonuclease reaction：

Nucleotide sequence (the SEQ ID NO of expression rEPIS albumen prepared by embodiment 1：16), the PCR of OmpK genes Product and pET-32a (+) (article No.：69015-3；Producer：Novagen double digestion) is carried out respectively, and endonuclease reaction system is： 10 4.5 μ L, BamHI and HindIII (article No.s of × K buffer：1010S and 1060S, producer：Precious day doctor's biotechnology (Beijing) has Limit company) each 1.5 μ L, express the nucleotide sequence of rEPIS albumen or the PCR product or pET-32a (+) plasmid of OmpK genes (two kinds of PCR product final concentrations are 20.0 μ L：25ng/μL；Plasmid is final concentration of：32ng/ μ L), 2.5 μ L of ddH2O are overall System is 30.0 μ l.37 DEG C of water-baths are stayed overnight, and digestion products are detected with 0.8% agarose gel electrophoresis.It is returned using DNA gel Receive kit (article No.：DP208；Producer：TIANGEN Biotech (Beijing) Co., Ltd.) recovery purifying double digestion product (volume It is 30 μ L).

2. connection reaction：

Double digestion product in step 1 is prepared into plasmid respectively.

Prepare pET-32a-repis expression plasmids：10 × T4 buffer (article No.s：2011A, producer：Bao doctor's object skill Art (Beijing) Co., Ltd) 2.5 μ L, T4 ligase (article No.s：2011A, producer：Precious day cures biotechnology (Beijing) limited public affairs Department, final concentration of 14U/ μ L) 1.0 μ L, pET-32a (+) double digestion product 14.5 μ L, rEPIS albumen nucleotide sequence 7.0 μ L of double digestion product, 25.0 μ L of total volume, after each ingredient mixing, overnight in 16 DEG C of water-baths connections.

Prepare pET-32a-OmpK expression plasmids：10×T₄Buffer (article No.s：2011A, producer：Precious day cures biotechnology (Beijing) Co., Ltd) 2.5 μ L, T₄Ligase (article No.：2011A, producer：Precious day cures biotechnology (Beijing) Co., Ltd, Final concentration of 14U/ μ L) 1.0 μ L, pET-32a (+) 14.5 μ L, OmpK genes of double digestion product PCR product double digestion 7.0 μ L of product, 25.0 μ L of total volume, after each ingredient mixing, overnight in 16 DEG C of water-baths connections.

The preparation of 3 polypeptides of embodiment/albumen

The preparation of the OmpK epitope polypeptides (repis) of 1 recombination

(1) conversion of recombinant expression plasmid

Reference《Molecular Cloning:A Laboratory guide》In method prepare E.coli Rosetta (DE3) competent cell, take 2 pipes 100 μ L E.coli Rosetta (DE3) competent cells, wherein a pipe competent cell is coated with LB agar plates as a contrast Detect cell activity；The recombinant plasmid pET-32a- that 20ng embodiments 2 are prepared is added in another pipe competent cell Repis, after mixing ice bath 30min；After 42 DEG C of water-bath thermal shock 1min, it is put into ice bath acts on 1min rapidly；Sterile item LB culture solutions shaken cultivation (37 DEG C, 160r/min) 60min that 800 μ L are free of Amp is added under part；5,000r/min is centrifuged 3min abandons supernatant, adds 200 μ L LB culture solutions and precipitation is resuspended, 100 μ L culture solutions is therefrom taken to be coated on the LB fine jades containing Amp Fat tablet, 37 DEG C of culture 12-16h, obtains the recombinant bacterium pET-32a- for having converted recombinant expression plasmid pET-32a-repis The bacterium colony of repis/Rosetta (DE3).

(2) identification of recombinant expression plasmid

The single bacterium colony that recombinant expression plasmid pET-32a-repis has been converted in picking (1) is cultivated, according to plasmid purification Kit (article No.：DP103；Producer：TIANGEN Biotech (Beijing) Co., Ltd.) specification extraction purification recombinant expression plasmid PET-32a-repis (amplification template) carries out recombinant expression plasmid pET-32a-repis after purification using P1, P2 as primer PCR is identified.Agarose gel electrophoresis, which is shown in 216bp, nearby the DNA bands of a specificity (see Fig. 6)；Sequencing result is shown The exogenous gene sequence of connection is identical with the multi-epitope of design series connection peptide gene sequence, shows recombinant plasmid pET-32a- Repis is transformed into E.coli Rosetta (DE3) successes.

In above-mentioned PCR identifications, PCR reaction systems are as shown in table 7；The sense primer P1 of design is 5 '- ATGGATATCCACAAAAACGAT-3′(SEQ ID NO：19), downstream primer P2 is 5 '- AGATTCTGGTTTGTGACCAGAACC-3′(SEQ ID NO：20)；PCR reaction conditions are：95 DEG C of pre-degeneration 4min；94℃ It is denaturalized 1min, 50 DEG C of annealing 30s, 72 DEG C of renaturation 30s, totally 25 cycles；Extend 10min after 72 DEG C.It is converted into work(simultaneously PCR positive plasmids deliver Shanghai biotechnology Services Co., Ltd and carry out sequencing identification.

Table 7：PCR reaction systems (50.0 μ L)

(3) induced expression

The single bacterium that recombinant expression plasmid pET-32a-repis has been converted in picking (1) falls within 5mL containing 100 μ g/mL Amp In LB fluid nutrient mediums (Amp+Cl resistances), 37 DEG C, 200rpm continues culture 12-14 hours.According to 1:100, take 2mL bacterium solutions It is added in 200mL LB culture mediums (Amp+Cl resistances) and expands culture, 37 DEG C, 200rpm, shake culture about 2-3h, until When OD600=0.4-0.5, it is extremely final concentration of that derivant IPTG (isopropylthiogalactoside) is added in culture 1mmol/L.After IPTG is added, flask is put into low temperature shaking table at once, 16 DEG C, 80rpm, continues culture 16 hours.Culture After 16h, 4 DEG C, 10000rpm centrifuges 5min, discards culture medium, collects bacterial sediment.

(4) identification of expression product and expression-form analysis

By the above-mentioned thalline being collected into 20mL sterilizing PBS (Na₂HPO₄·12H₂O 2.9g, KH₂PO₄0.2g, NaCl 8.0g, KCl 0.2g, add distilled water to 1000mL, and pH to 7.4 is adjusted with NaOH solids.121 DEG C sterilize 20 minutes) it is resuspended, make Thalline is obtained to be resuspended in buffer solution.It is resuspended and is precipitated with lysis buffer, PMSF (phenylmethylsulfonyl fluoride) is added, and (working concentration is 1mM) carries out ultrasonic cracking afterwards three times with -20 DEG C of placements of lysozyme (working concentration 0.2-0.4mg/ml), multigelation and (adopt With 300W, ultrasonic 4s, stop 8s, amount to 20min), keep thalline fully broken.4 DEG C, 10000rpm, 20min of sample after ultrasound, Precipitation and separation and supernatant, supernatant are kept separately precipitation and are resuspended with the PBS of same volume 20mL, and supernatant is solubility portion Point, it is precipitated as forgiving body portion.Precipitation and supernatant respectively take 8uL, are separately added into 5 × SDS-PAGE sample-loading buffers of 2uL, boil 10min is boiled, loading carries out SDS-PAGE detections (12% separation gel, 5% concentration glue, 80V voltage stabilizings electrophoresis about 20min to bromophenol blue Into separation gel, voltage rises to 120V, and electrophoresis 70min to bromophenol blue reaches gel bottom).Remove gel Coomassie brilliant blue Dyeing liquor boils dyeing 2min, discards dye liquor；By glue as boiling decoloring 20min in clear water, water is during which changed 2 times.

(5) purifying of rEPIS recombinant proteins

The recombinant protein of solubility expression, by supernatant and Ni-binding buffer (equilibration buffer) according to 1:1 is mixed After conjunction, upper Ni-His resin affinity chromatographic columns carry out affinity purification；The albumen of inclusion body expression is straight by the inclusion body of collection It connects and is purified after being resuspended with 20 mLNi-binding buffer (equilibration buffer).Ni-His resin are using preceding flowing to end guarantor Liquid is protected, is then fully washed with 30 times of Ni-His resin volumetric balances buffer solutions.Mixed protein solution and filler dynamic In conjunction with after having flowed, the washing buffer of 30 times of Ni-His resin column volumes is added, fully in coutroi velocity 0.5mL/min Washing.With the elution buffer of 5-10mL, Ni-His resin are added, after acting on 10min, coutroi velocity is received in 1mL/min Collect the destination protein of elution.The destination protein of elution is concentrated by ultrafiltration pipe using 3KD and concentrates, with PBS displacing elution liquid, last mesh Protein dissolution in PBS, measured concentration, -80 DEG C freeze it is spare.

2 label proteins, the preparation for recombinating OmpK albumen

Expression plasmid pET-32a-OmpK and pET-32a are converted according to 1 (1) conversion condition to competent cell respectively E.coli Rosetta (DE3) carry out prokaryotic expression.Random picking conversion is flat after expression plasmid pET-32a-OmpK conversion cultures Single bacterium colony on plate is cultivated, and extraction recombinant plasmid carries out PCR identifications, identifies that agarose gel electrophoresis is shown in by PCR 759bp nearby has the DNA bands (see Fig. 7) of a specificity, PCR to identify that the primer is SEQ ID NO：17 and SEQ ID NO：Sequence shown in 18, PCR system and condition are configured and are reacted according to system and condition shown in 1 (2).PCR product passes through Sequencing is accredited as OmpK sequences.According to 1 (3) -1 (5) expression condition carry out great expression, purifying, preserve recombination OmpK albumen and Label protein His-tag.

The identification of 3 albumen

In gene engineering recombinant bacterium pET-32a-repis/Rosetta (DE3), pET-32a-OmpK/Rosetta (DE3) And after IPTG is added in pET-32a/Rosetta (DE3), after 16 DEG C of Fiber differentiation 16h, thalline were collected by centrifugation, will be upper after ultrasound Clear liquid and precipitation are respectively through SDS-PAGE electrophoresis, the precipitation of pET-32a-OmpK/Rosetta (DE3) after induction, through SDS- There is dense dye protein band (Fig. 8, swimming lane 1) in the positions 47KD in PAGE electrophoresis, meets expected recombination OmpK molecular weight of albumen, There is no this protein band (Fig. 8, swimming lane 2) in supernatant, it is inclusion body egg to illustrate that the inductive condition descends the recombination OmpK albumen of expression In vain；In the supernatant of pET-32a-repis/Rosetta (DE3) after induction, there is dense dye albumen in the left and right positions 30kDa Band (Fig. 8, swimming lane 8), meet it is expected recombinate rEPIS molecular weight of albumen, do not have in precipitation the dense dye protein band (Fig. 8, Swimming lane 9), illustrate that the recombination rEPIS albumen expressed under the inductive condition is soluble protein；PET-32a/ after induction The label protein (Fig. 9, swimming lane 5) that a size is about 20.4kDa is shown after Rosetta (DE3) supernatant electrophoresis.

The recombination rEPIS albumen and label protein of solubility expression, by supernatant Ni-His resin affinity chromatographic columns Carry out affinity purification；The recombination OmpK albumen of inclusion body expression, by the inclusion body of collection Ni-His resin affinity chromatographic columns Carry out affinity purification.SDS-PAGE electrophoresis is carried out after purification, detects destination protein (Fig. 9) after purification.

Embodiment 4：Immunologic specificity detects

1. vibrio parahaemolytious ATCC17802 measures kunming mice LD50

Picking vibrio parahemolyticus ATCC17802 bacterial strain single bacterium colonies are inoculated in the training of 100mL3%NaCl alkaline peptone water bodies It supports in base, for 24 hours, 8000r/min is centrifuged 8 minutes 30 DEG C of shaken cultivations, collects thalline, is resuspended with appropriate sterile PBS, and dilute At 1 × 10¹⁰、2×10⁹、4×10⁸、8×10⁷、1.6×10⁷It is dense to calculate bacterium with Maxwell opacity tube for 5 gradients of CFU/mL Degree.

Experiment kunming mice (50g) is divided into 6 groups, every group 11,1-5 groups are experimental group, and the 6th group is PBS control group. The bacterium solution of above-mentioned 5 dilutions is injected intraperitoneally in 1-5 groups respectively, and only, the sterile PBS of equivalent is injected intraperitoneally in control group to 0.1mL/.Observation Viscera situation is observed in mouse invasion and death condition in record 14 days, the sterile dissection of the mouse that dies of illness, and picking internal organ are inoculated with In separation of bacterial again on thiosulfate-citrate-cholate-sucrose (TCBS) agar medium.

Experimental mice attacks poison with 5 dilution bacterial suspensions respectively, and mouse survival and death condition are shown in Table 8 in 2 weeks, root LD of ATCC17802 plants of the vibrio parahaemolytious to kunming mice is calculated according to Reed-Muench methods₅₀It is 3.85 × 10⁷CFU/mL。 lgLD₅₀=higher than 50% death rate dilution inverse logarithm+distance proportion × extension rate logarithm, distance proportion=(height In 50% death rate -50)/(being less than 50% death rate higher than 50% death rate -).

Table 8：ATCC17802 plants of vibrio parahaemolytious survives to kunming mice and death condition statistical form

2. the preparation of Commercial bacterin

Vibrio parahemolyticus ATCC17802 bacterial strain single bacterium colonies are seeded to 150mL3%NaCl basic protein peptone culture solutions, 30 DEG C of shaken cultivations are for 24 hours.Thalline are inactivated in 37 DEG C of oscillations for 24 hours, be centrifuged repeatedly with sterile PBS using final concentration of 0.3% formaldehyde 6 times, formaldehyde is removed, thalline is resuspended to 1 × 10 with sterile PBS⁹CFU/mL.200 μ L bacterium solutions are taken to be coated with TCBS solid plates, inspection Look into inactivating efficacy.

3. kunming mice is grouped and immune programme

Kunming mice is randomly divided into 5 groups, and 1-4 groups are attached most importance to respectively, and (injection rEPIS albumen and Freund are incomplete for a group rEPIS groups Adjuvant // Freund's complete adjuvant), (injection recombination OmpK albumen and incomplete Freund's adjuvant/Freund are complete for recombination OmpK protein groups Adjuvant), inactivated vaccine group (injection inactivated vaccine) and His-Taq groups (inject His-Taq label proteins and Freund not exclusively helped Agent/Freund's complete adjuvant), the 5th group is PBS control group (injection PBS solution), every group 20.The immunizing dose of 3 kinds of albumen is 2 μ g/g weight, when first immunisation, the immunizing dose of inactivated vaccine is 1 × 10⁸Only, immunization route is intraperitoneal injection, egg to CFU/ In vain with incomplete Freund's adjuvant according to volume ratio 1:1 mixing and emulsifying.Booster immunization is primary after 2 weeks, when booster immunization, albumen with Freund's complete adjuvant is according to volume ratio 1:1 mixing and emulsifying, dosage is identical as initial immunity, and control group injects equivalent PBS.

4. immunized mice antibody level of serum detects

The 14th day and the 28th day after first immunisation, takes a blood sample from each group immunized mice eye socket and detach serum, respectively with the weight of purifying Group rEPIS albumen, recombination OmpK albumen and label protein His-Taq are envelope antigen, are measured using indirect ELISA method special Heterogenetic antibody is horizontal.As a result display is as shown in table 9, and recombination rEPIS albumen and recombination OmpK albumen can stimulate body well Generate the antibody of higher level.Label protein His-Taq can also stimulate body to generate corresponding antibodies, but its antibody level is remote Horizontal far below anti-recombination rEPIS albumen and recombination OmpK protein antibodies, i.e. antibody in two groups of immune serums is with anti-recombination Based on rEPIS albumen, recombination OmpK albumen.

Concrete operations are as follows：

(1) respectively with the recombination rEPIS albumen of a concentration of 1 μ g/ml, recombination OmpK albumen and label protein His-Taq packets By enzyme mark hole, 100 holes μ l/, capping is placed in metal wet box and stays overnight for 4 DEG C.

(2) hole endoperidium liquid is discarded, is washed 5 times with pH 7.4PBST, 1min is vibrated on oscillator, is patted dry on paper.

(3) with the pH7.4PBS solution containing 5% skimmed milk power, 37 DEG C of closing 2h.

(4) discard confining liquid, washed by step (2) method, be added it is immune after different time and different dilutions it is to be checked Blank control, 37 DEG C of reaction 1h are in serum and negative serum, 100 holes μ l/, last hole.

(5) washing methods is added 1 with (2):7000 diluted sheep anti mouse HRP-IgG, 100 holes μ l/, 37 DEG C of reaction 1h.

(6) 100 μ l of o-phenylenediamine (OPD) substrate solution are added per hole with (2) for washing methods, and room temperature, which is protected from light, stands 10min, Add 50 μ l of terminate liquid per hole, each hole OD492 values are detected in microplate reader after gently shaking.With serum OD (P)/negative serum to be checked OD (N) >=2.1 is judged as the positive, and measures antibody titer.

Table 9：Antibody level ELISA testing results in kunming mice Post-immunisation serum

5. recombinating rEPIS protein immunization reactivity

Purifying recombination rEPIS albumen is carried out carrying out Western-blot analyses, analyzes its immunoreactivity.Purifying recombination REPIS albumen first carries out SDS-PAGE, and Western-blot analyses are carried out after electrophoresis.To recombinate rEPIS protein immunization groups The antiserum of 28d mouse orbits blood sampling separation is primary antibody after first immunisation, is carried out to purifying recombination rEPIS albumen Western-blot is analyzed, and as a result shows (Figure 10), and the recombinant protein rEPIS of purifying can occur with the anti-recombination rEPIS antibody of mouse , there is a specific immune response band at 30kDa in specific binding reaction.

Concrete operation step is as follows：

(1) pvdf membrane and 6 filter paper are cut according to the size of separation gel.Pvdf membrane infiltrates in absolute ethyl alcohol, then spends Film is put into protein delivery electrophoresis liquid and impregnates by ionized water rinse, and filter paper and transfer pad are impregnated using preceding in shifting liquid.

(2) according to the suitable of cathode--3 layers of transfer pad filter paper-SDS-PAGE glue--3 layers of pvdf membrane filter paper-transfer pad-anode Sequence is put well, and bubble is driven away with glass bar, is put into transfer groove, addition electrophoretic blotting liquid is top to dipped transfer blade, is put in ice chest Enter ice cube, 100V voltage stabilizing transferring films 1-2h.

(3) it takes the film out and is put into the PBS confining liquids containing 5% skimmed milk power, 4 DEG C of closings are overnight.

(4) it taking the film out and is put into clean plate, elder generation wash with distilled water, then with PBST washs, and shakes washing 3 times, 10min/ times, the antiserum detached is taken a blood sample as primary antibody using 28d mouse orbits after recombinating rEPIS protein immunization group first immunisations, is pressed According to 1:It is added after 7500 dilutions, is slowly shaken on shaking table and be incubated 1h.

(5) primary antibody is exhausted, PBST cleaning solutions is added to shake washing 3 times, 10min/ times.

(6) it exhausts cleaning solution and is added 1:5000 diluted sheep anti mouse HRP-IgG, room temperature, which is slowly shaken, is incubated 1h.Exhaust enzyme Secondary antibody is marked, PBST cleaning solutions is added to shake washing 3 times, 10min/ times.

(7) DAB substrate solutions colour developing about 15min is added, adds distilled water color development stopping.By the result and the pre-dyed albumen of developing the color Molecular criteria is compared.

6. recombinating rEPIS protein immunization protectiveness to measure

Each experimental mice 30th day after immune, respectively with 10LD50 vibrio parahemolyticus ATCC17802 plants attacked Poison experiment, as a result using immune relative protection ratio (Relative percent survival, RPS)=(1- immune group death Rate/control group the death rate) × 100% calculation calculate relative protection ratio, as a result, it has been found that label protein group and PBS control group 20 mouse are all dead, and recombination rEPIS group grass carps survive 18, relative protection ratio 90%；Recombinate OmpK group mouse survivals 17, relative protection ratio 85%；Inactivated bacteria group mouse survival 18, relative protection ratio are 90% (being shown in Table 10).Dead mouse abdomen Portion's swelling, visible liver enlargement and colour changed into yellow after dissect have yellow hydrops in enteron aisle, wiping tablet, 4th area are applied with liver section Scribing line separation of bacterial, is identified as vibrio parahemolyticus.

Table 10：Test mice immune protective test result

From the above it is found that successful design of the present invention, expression and having purified vibrio parahemolyticus OmpK multilist bit serials Peptide rEPIS, tandem polypeptide rEPIS can induce kunming mice to generate the specific antibody of higher level, and can protect 90% kunming mice resists the infection of 10LD50 vibrio parahemolyticus, has good immunoprotective effec.

Embodiment 5：Kit and its application

It present embodiments provides a kind of kit and its application, the kit includes：

a)SEQ NO ID:Polypeptide or derivatives thereof shown in 15；

B) adjuvant：Incomplete Freund's adjuvant, Freund's complete adjuvant.

The experiment proved that the kit has the characteristics that safe and efficient, its immunogene very high to vibrio parahemolyticus tool Property, the animal through this multi-epitope polypeptide immune, the antibody in vivo containing a large amount of specificity, to immune animal Effective Vate of Protection Reach 90% or more.The multi-epitope polypeptide, which improves, to be received the animal of this multi-epitope polypeptide immune and is supported to vibrio parahemolyticus Anti- ability has good immunoprotective effec.

Mentioned reagent box can be used for prevention and control aquatic livestock infectious disease.

The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen Specific implementation please is confined to these explanations.For those of ordinary skill in the art to which this application belongs, it is not taking off Under the premise of conceiving from the application, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the protection of the application Range.

SEQUENCE LISTING

<110>Shantou Entry-Exit Inspection and Quarantine Bureau, People's Republic of China

<120>A kind of OmpK multi-epitopes polypeptide, construction method and its application of recombination

<130> 201800305

<160> 33

<170> PatentIn version 3.3

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gatatccaca aaaacgatta c 21

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aacgagaaag gttatgctga atcttctcat gattac 36

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ccaggcagcg acaaagctgg c 21

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tacgatggca acaagaaaga ttgg 24

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gatgacgaca aaggtaacaa g 21

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ggtcacaaac cagaatct 18

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ggtggtggtt ct 12

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Asp Ile His Lys Asn Asp Tyr

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Asn Glu Lys Gly Tyr Ala Glu Ser Ser His Asp Tyr

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Pro Gly Ser Asp Lys Ala Gly

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Tyr Asp Gly Asn Lys Lys Asp Trp

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Asp Asp Asp Lys Gly Asn Lys

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Gly His Lys Pro Glu Ser

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Gly Gly Gly Ser

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Asp Ile His Lys Asn Asp Tyr Gly Gly Gly Ser Asn Glu Lys Gly Tyr

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

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Ala Gly Gly Gly Gly Ser Tyr Asp Gly Asn Lys Lys Asp Trp Gly Gly

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Gly Ser Asp Asp Asp Lys Gly Asn Lys Gly Gly Gly Ser Gly His Lys

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Pro Glu Ser

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cgcggatcca tggatatcca caaaaacgat tacggtggtg gttctaacga gaaaggttat 60

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ggttcttacg atggcaacaa gaaagattgg ggtggtggtt ctgatgacga caaaggtaac 180

aagggtggtg gttctggtca caaaccagaa tctaagcttg gg 222

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cgggatccgc agattactct gacggcgata t 31

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cccaagcttt tagaatccgt aagttactgc ga 32

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atggatatcc acaaaaacga t 21

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agattctggt ttgtgaccag aacc 24

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

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Asp Glu Leu Pro Gly Glu Ser Ser His Asp Tyr

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

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Pro Gly Ser Asp Lys Ser Gly

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Lys Gly Ser Asp Lys Asn Gly

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Tyr Asp Ser Asn Lys Lys Asp Trp

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Tyr Asp Ser Asn Gln Lys Asp Trp

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Tyr Tyr Gly Asn Asn Lys Asp Trp

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Asp Asp Asp Ala Gly Asn Lys

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Glu Asp Lys Glu Gly Asn Lys

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Ala Gln Thr Ala Asp Phe

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Thr Gln Thr Val Asp Ser

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Asn Glu Leu Pro Gly Phe Pro Asp Gly Ser Asn His Asp Tyr

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Claims

1. OmpK multi-epitope polypeptides of a kind of recombination or derivatives thereof, which is characterized in that described polypeptide or derivatives thereof is selected from：

2) the amino acid fragment sequence for including has at least one or several amino acid in 1) respectively compared with amino acid fragment Replace, lack and or add, the polypeptide has the immunogenicity to vibrios；

4) derivative of polypeptide is 1) -3) derivative of any polypeptide, the derivative of the polypeptide has to vibrios Immunogenicity.

2. polypeptide according to claim 1 or derivatives thereof, which is characterized in that also include joint sequence 1, the connector sequence Row 1 are between amino acid fragment sequence；

Preferably, the joint sequence 1 is SEQ ID NO：Sequence shown in 14；

Preferably, the amino acid fragment sequence is according to SEQ ID NO：8-SEQ ID NO：13 are sequentially connected with；

Preferably, the polypeptide is SEQ ID NO：Sequence shown in 15.

3. a kind of OmpK nucleotide of recombination, which is characterized in that the nucleotide is used for encoding any rights of claim 1-2 It is required that the polypeptide or derivatives thereof；

Preferably, coding SEQ ID NO：8-SEQ ID NO：The nucleotide fragments of amino acid fragment sequence shown in 13 are respectively SEQ ID NO：1-SEQ ID NO：Nucleotide sequence shown in 6, also includes joint sequence 2, and the joint sequence 2 is located at nucleosides Between acid sequence；

Preferably, the joint sequence 2 is SEQ ID NO：It is nucleotide sequence coded shown in 7；

Preferably, the nucleotide fragments are according to SEQ ID NO：1-SEQ ID NO：7-SEQ ID NO：2-SEQ ID NO： 7-SEQ ID NO：3-SEQ ID NO：7-SEQ ID NO：4-SEQ ID NO：7-SEQ ID NO：5-SEQ ID NO：7-SEQ ID NO：It is sequentially connected with shown in 6.

4. nucleotide according to claim 3, which is characterized in that the end of nucleotide 5 ' further includes initiation codon Subsequence；

Preferably, the initiator sequences are ATG；

Preferably, 5 ' ends of the initiator sequences further include cleavage sequence；

Preferably, the cleavage sequence at 5 ' ends of the initiator sequences is I restriction endonuclease recognition sequences of BamH；

Preferably, the 3 ' ends of the termination codon subsequence further include cleavage sequence；

Preferably, the cleavage sequence at the 3 ' ends of the termination codon subsequence is III restriction endonuclease recognition sequences of Hind；

Preferably, the nucleotide also includes protected nucleoside acid sequence；

Preferably, the protected nucleoside acid sequence is CGC and GGG；

Preferably, the sequence of the nucleotide is SEQ ID NO：Sequence shown in 16.

5. a kind of carrier, which is characterized in that the carrier includes the nucleotide described in claim 3-4 any claims；

Preferably, the carrier is pET-32a-SEQ ID NO：16.

6. a kind of cell, which is characterized in that the cell contains the carrier described in claim 5；

Preferably, the cell is pET-32a-SEQ ID NO：16/E.coli Rosetta cells.

7. a kind of vaccine, which is characterized in that the vaccine include claim 1-2 any claims described in epitope polypeptide or Its derivative；

Preferably, the vaccine also includes adjuvant.

8. a kind of kit, the kit includes polypeptide described in claim 1-2 any claims or derivatives thereof, power Profit requires the carrier described in 5, the cell described in claim 6 and/or the vaccine described in claim 7.

9. nucleotide, power described in polypeptide described in claim 1-2 any claims or derivatives thereof, claim 3-4 Profit requires the carrier described in 5, the cell described in claim 6 and/or the vaccine described in claim 7 to prevent or control preparing Treat the application in the product of vibrio infection；

Preferably, the vibrios is vibrio parahemolyticus.

10. a kind of method of OmpK multi-epitope polypeptides or derivatives thereof of structure recombination, which is characterized in that the method includes Following steps：

(2) according to linear epitope, corresponding nucleotide fragments is designed and its are put in order, and design segment jointing, digestion Site, protected nueleotide, initiation codon, the position relationship of terminator codon and particular sequence；

(3) nucleotide sequence designed in (2) is obtained；

(4) polypeptide of (3) nucleotide sequence coding is obtained；

Preferably, the polypeptide in the step (4) is obtained by following steps：A. nucleotide step (3) obtained and expression Carrier connects；B. conversion carrier obtains the polypeptide of expression；

Preferably, the nucleotides sequence of the step (3) is classified as nucleotide sequence shown in claim 4；

Preferably, the nucleotides sequence of the step (3) is classified as SEQ ID NO:Sequence shown in 16；

Preferably, the polypeptide sequence of the step (4) is the polypeptide described in claim 1-2 any claims；

Preferably, the polypeptide sequence of the step (4) is SEQ ID NO:Sequence shown in 15；

Preferably, the expression vector is pET-32a (+)；

Preferably, the software is DNAstar protean softwares, be used for the secondary structure of comprehensive analysis OmpK albumen, flexibility, Surface possibility, hydrophily and antigenic index.