Vibrio parahaemolyticus tunica externa protein ompK subunit vaccine and preparation method thereof

A subunit vaccine, the technology of Vibrio hemolyticus, which is applied in the field of Vibrio parahaemolyticus outer membrane protein ompK subunit vaccine and preparation thereof, can solve problems such as loss, and achieve the effects of retaining immunogenicity and low preparation cost

Inactive Publication Date: 2008-05-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of aquaculture production, due to the strict detection of antibiotic residues in exported aquatic products and the prohibition of most antibiotics by the Department of F

Method used

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  • Vibrio parahaemolyticus tunica externa protein ompK subunit vaccine and preparation method thereof
  • Vibrio parahaemolyticus tunica externa protein ompK subunit vaccine and preparation method thereof
  • Vibrio parahaemolyticus tunica externa protein ompK subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] This example describes the method for obtaining the ompK gene of Vibrio parahaemolyticus zj2003 strain provided by the present invention, including the following steps:

[0061] (1) Preparation of Vibrio parahaemolyticus genomic DNA

[0062] Take a branch of Vibrio parahaemolyticus glycerol that was frozen at ultra-low temperature, inoculate it in Zobell 2216E medium after thawing at room temperature, and culture it with shaking at 28°C for more than 12 hours; take 1.5mL of the bacterial liquid in an Ependoff centrifuge tube, centrifuge at 5000r / min for 1min, discard supernatant; sterilized ddH 2 O to suspend the bacteria, centrifuge at 6000r / min for 4min, wash twice; add 35μLddH 2 O and 35μL TZ solution, placed at -20°C for 30-40min; boiling water bath for 10min; ice bath for 10min; 5000r / min, centrifuged for 5min, and the supernatant was collected as a PCR template. Formula of TZ solution: 4% Triton X-100, 5.0g / L NaN 3 , 25mmol / L Tris-HCl, pH8.0.

[0063] (2) PCR ...

Embodiment 2

[0072] This example describes the method for obtaining the ompK mature peptide coding sequence prokaryotic expression plasmid pET30a-ompK, the steps of which include:

[0073] (1) Amplification of OmpK mature peptide coding sequence

[0074] The signal peptide cleavage site of the ompK protein is predicted by the online software SignalP to be between the 20th and 21st amino acid residues at the N-terminus, and the mature peptide coding sequence is from the 61st nucleotide sequence after the start codon to the Stop codon, redesign primers at both ends (full length 759bp, encoding 253 amino acids)

[0075] P3 (5'-CG GGATCC GCAGATTACTCTGACGGCGATAT-3')

[0076] P4(5'-CCC AAGCTT TTAGAACTTGTAAGTTACTGCGA-3')

[0077] Both ends of the primers were respectively introduced with enzyme cutting sites BamHI (underlined at P3) and HindIII (underlined at P4), and the primers were synthesized at Shanghai Yingjun Biotechnology Co., Ltd. Using T-ompK as template and P3 and P4 as primers, ...

Embodiment 3

[0082] This example describes the method for obtaining ompK protein. Include the following steps:

[0083] (1) Expression of OmpK mature peptide coding sequence prokaryotic expression plasmid pET30a-ompK in Escherichia coli

[0084] Transformation of recombinant plasmid pET30a-ompK into CaCl 2 Escherichia coli BL21(DE3) prepared by the method, and the identified positive clone pET 30a-ompK was cultured in LB liquid medium containing kanamycin (50ug / ml) until the OD value reached 0.5-0.6, and IPTG was added to the final concentration 1.0mmol / L, induce expression at 37°C for 3-4 hours, collect bacterial cells by centrifugation, and partially lyse the bacterial cells in an ice bath with a power of 300W until the solution becomes translucent bacteria, centrifuge at 1500×g for 30min, and collect Supernatant and precipitate: Take bacterial cells, precipitate and supernatant after ultrasonic disruption of bacterial cells, add 5× loading buffer, boil at 100°C for 5 minutes, and carr...

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Abstract

The invention discloses protein ompK subunit vaccine of assistant haemolysis vibrio extine and a preparation method thereof. The vaccine is PBS solution which converts the recombination protein of the coliform bacteria of recombination prokaryon expression plasmid pET30a-ompK expressed by inducing and after being purified; the concentration of the PBS solution is 0.25-0.5mg/ml. The method has the steps that: firstly, the extraction of an assistant haemolysis vibrio full gene group, the overall length of extine protein ompK DNA and the clone of a mature peptide coded sequence; secondly, the construction of a prokaryon expression plasmid of the ompK mature peptide coded sequence, thirdly, the obtaining way of the recombination ompK protein; fourthly, the detection to the immunity way and the immunity effect of a large yellow croaker with recombination ompK protein. The invention provides the preparation method of the assistant haemolysis vibrio ompK protein subunit vaccine, and simultaneously provides the detection method of the immunity effect of the large yellow croaker with recombination ompK protein, the preparation method is simple, and the usage is convenient.

Description

technical field [0001] The invention relates to a vibrio parahaemolyticus outer membrane protein ompK subunit vaccine and a preparation method thereof. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) initially attracted people's attention because it is an important human intestinal pathogen. It is often caused by ingestion of uncooked seafood, causing symptoms such as vomiting and diarrhea. In recent years, a variety of seawater economic animals cultured in China have been harmed by the bacteria, including large yellow croaker, flounder, prawns, crabs and cultured shellfish, etc., resulting in serious losses. At present, it is generally believed that the bacterium is a common bacterium in the seawater environment and an opportunistic pathogen of farmed animals. The immunogenicity of the main outer membrane protein of this bacterium has been observed in earlier studies, but because humans can effectively prevent the infection of this bacterium...

Claims

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Application Information

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IPC IPC(8): A61K39/106C12N15/31C12N15/63C12N15/70A61P31/04A61P1/08A61P1/10
Inventor 于涟毛芝娟由振强张祟文魏永伟
Owner ZHEJIANG UNIV
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