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Recombinant OmpK multi-epitope polypeptide, construction method and application thereof

A multi-epitope, 8-SEQIDNO technology, applied in the field of biomedicine, can solve the problems of poor immune effect and large side effects, and achieve the effect of improving resistance, high immunogenicity and reducing mortality

Active Publication Date: 2018-09-14
中华人民共和国汕头海关
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although inactivated vaccines are very safe, they have the disadvantages of poor immune effect and large side effects

Method used

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  • Recombinant OmpK multi-epitope polypeptide, construction method and application thereof
  • Recombinant OmpK multi-epitope polypeptide, construction method and application thereof
  • Recombinant OmpK multi-epitope polypeptide, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 prepares nucleotide sequence

[0073] 1 Design and synthesis of OmpK protein epitope tandem gene

[0074] (1) Selection of OmpK protein amino acid sequence

[0075] Log in the nucleotide sequence and amino acid sequence of the Vibrio parahaemolyticus OmpK gene searched in the National Center for Biological Information (NCBI) of the United States. After searching, there are 13 nucleotide sequences of the Vibrio parahaemolyticus OmpK registered by NCBI, such as As shown in Table 1, the length of the open reading frame (ORF) ranges from 792 to 822 bp, encoding 264 to 274 amino acids, and the molecular weight of OmpK protein is between 29.4KD to 30.5KD. The gene sequence accession number of the OmpK protein of ATCC17802 is HM044386.1 , ORF length is 819bp, encoding 273 amino acids, OmpK protein molecular weight is 30.3KDa. Using DNAStar Megalign software, the nucleotide and amino acid sequences of 13 OmpK genes were homologously compared and analyzed, and the...

Embodiment 2

[0100] Example 2 Preparation of expression plasmid

[0101] 1. Enzyme digestion reaction:

[0102] The nucleotide sequence (SEQ ID NO: 16) of the expressed rEPIS protein prepared in Example 1, the PCR product of the OmpK gene, and pET-32a(+) (product number: 69015-3; manufacturer: Novagen) were subjected to double enzyme digestion respectively , the enzyme digestion reaction system is: 4.5 μL of 10×K buffer, 1.5 μL each of BamHI and HindIII (Cat. No.: 1010S and 1060S, manufacturer: Baoriji Biotechnology (Beijing) Co., Ltd.), expressing the nucleotide sequence of rEPIS protein or OmpK gene PCR product or pET-32a(+) plasmid 20.0 μL (final concentration of both PCR products: 25ng / μL; plasmid final concentration: 32ng / μL), ddH2O 2.5 μL, total system 30.0 μl. React overnight in a water bath at 37°C, and use 0.8% agarose gel electrophoresis to detect the digested products. DNA gel recovery kit (article number: DP208; manufacturer: Tiangen Biochemical Technology (Beijing) Co., Ltd....

Embodiment 3

[0107] The preparation of embodiment 3 polypeptide / protein

[0108] 1 Preparation of recombinant OmpK epitope polypeptide (repis)

[0109] (1) Transformation of recombinant expression plasmids

[0110] Prepare E.coli Rosetta (DE3) competent cells according to the method in "Molecular Cloning Experiment Guide", take 2 tubes of 100 μL E.coli Rosetta (DE3) competent cells, and one of the tubes of competent cells is used as a control to coat LB agar plates Detect cell activity; add 20ng of the recombinant plasmid pET-32a-repis prepared in Example 2 to another tube of competent cells, mix well and then ice-bath for 30 minutes; after heat-shocking in a water bath at 42°C for 1 minute, quickly place it in an ice-bath for 1 minute ; Under sterile conditions, add 800μL of LB culture medium without Amp and shake culture (37℃, 160r / min) for 60min; centrifuge at 5,000r / min for 3min, discard the supernatant, then add 200μL of LB culture medium to resuspend the pellet, and take 100μL from ...

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Abstract

The invention relates to the field of biomedicines and in particular relates to a recombinant polypeptide, a construction method and application thereof. More specifically, the invention provides a recombinant OmpK multi-epitope polypeptide and encoded nucleotide, carriers, cells and vaccines thereof, and application of the recombinant OmpK multi-epitope polypeptide to products of preventing vibrio infections. An experiment proves that the recombinant OmpK multi-epitope polypeptide or derivatives and vaccines thereof have the characteristics of safety and high efficiency and have very high immunogenicity on vibrio parahaemolyticus; animals immunized by the multi-epitope polypeptide contain a lot of specific antibodies and the effective protection rate on the immunized animals reach 90 percent or more; the recombinant OmpK multi-epitope polypeptide has the effect of improving the resistance capability of the animals immunized by the multi-epitope polypeptide on the vibrio parahaemolyticus; the recombinant OmpK multi-epitope polypeptide has a good immune protection effect and has great significance in the aspect of preventing and controlling infectious diseases of aquatic animals.

Description

technical field [0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to recombinant polypeptides, construction methods and applications thereof. More specifically, the present invention relates to recombinant polypeptides and their encoded nucleotides, vectors, cells, and vaccines in the prevention of Vibrio infection. use in the product. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus, VP) is a Gram-negative bacterium belonging to Vibrio family Vibrio, commonly known as halophilic bacteria, Vibrio parahaemolyticus, widely exists in the marine environment and river estuaries, and can infect fish It is one of the main pathogenic bacteria threatening the mariculture industry and has brought huge economic losses to the aquaculture industry. This bacterium infects as many as 48 kinds of marine fishes, has a high incidence rate, a wide range of prevalence, and serious harm, which is one of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/28C12N15/85C12N5/10C12N15/31A61K39/106A61P31/04
CPCA61K39/107A61P31/04C07K14/28C12N15/85
Inventor 张玉晴许如苏汪天杰张峥嵘吴松浩许晓升沈烨周铮宇
Owner 中华人民共和国汕头海关
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