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Crassostrea hongkongensis BPI gene, encoded protein and cloning method, and construction method for crassostrea hongkongensis BPI genetic engineering strain

A technology of genetically engineered bacteria and encoded proteins, applied in the field of biomolecular markers, can solve problems such as endangering ecological safety, drug residues, and inability to effectively control diseases, and achieve the effect of efficient killing

Inactive Publication Date: 2020-06-19
BEIBU GULF UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Blindness and abuse of antibiotics will not only fail to effectively control diseases, but will also damage the water environment and endanger ecological security at the same time
Long-term medication will lead to the resistance of pathogenic bacteria to drugs, and bring about a series of problems such as environmental pollution and drug residues in shellfish.

Method used

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  • Crassostrea hongkongensis BPI gene, encoded protein and cloning method, and construction method for crassostrea hongkongensis BPI genetic engineering strain
  • Crassostrea hongkongensis BPI gene, encoded protein and cloning method, and construction method for crassostrea hongkongensis BPI genetic engineering strain
  • Crassostrea hongkongensis BPI gene, encoded protein and cloning method, and construction method for crassostrea hongkongensis BPI genetic engineering strain

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Experimental program
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Effect test

Embodiment

[0043] 1. Cloning of BPI gene

[0044] 1.1 Primer design and synthesis

[0045] According to the gene sequence of the BPI gene of the homologous species published in GenBank, specific primers were designed using primer 5.0 software

[0046] Ch-BPI-F1: TGTTGTGTGAAAAGCCACA;

[0047] Ch-BPI-R1: TATATCCGTCTTCTGTGAAAAA.

[0048] 1.2 Total RNA extraction and first-strand cDNA synthesis

[0049] Total RNA was extracted from Hong Kong giant oyster by Trizol method. After the RNA was extracted, its purity and integrity were detected by spectrophotometer and electrophoresis, and stored at -80°C for future use. The reverse transcription reaction was performed according to the instructions of the AMV reverse transcription kit. Reverse transcription system 20 μl: Total RNA 2 μg, DNasel 1 μl, DNasel Buffer 1.3 μl, EDTA 1 μl, dNTP 1 μl, random primer 2 μl, 5×AMV Buffer 4 μl, RNase Inhibitor 0.5 μl, AMV reverse transcriptase 1 μl, RNase FreeH2O to 20 μl . Reaction conditions: 5min at 2...

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Abstract

The invention discloses a crassostrea hongkongensis BPI gene, an encoded protein and a cloning method therefor, and a construction method for a crassostrea hongkongensis BPI genetic engineering strainand application of a recombinant protein thereof. The sequence of the crassostrea hongkongensis BPI gene is shown by ESQ ID NO:1, and the cloning method comprises designing a specific primer amplified gen according to a conserved sequence isogenous to the BPI gene; a sequence of the crassostrea hongkongensis BPI protein is shown by SEQIDNO.2; primers containing an Ndel locus and an Xbal locus areconfigured to amplify a crassostrea hongkongensis BPI mature protein; and a target gene obtained by cloning is inserted into a carrier to obtain a recombinant plasmid, inducible expression is conducted on the recombinant plasmid, and the genetic engineering strain is obtained through purification and renaturation. The method has the advantage of having an obvious bactericidal effect on vibrio alginolyticus and vibrio parahaemolyticus.

Description

【Technical field】 [0001] The invention relates to the technical field of biomolecular markers, in particular to a Ch-BPI gene, a coded protein and a cloning method thereof, and a method for constructing a Ch-BPI genetic engineering bacterium of the Hong Kong giant oyster. 【Background technique】 [0002] Bactericidal permeability-increasing protein (BPI) is a member of the lipid transfer / lipopolysaccharide-binding protein family and plays a vital role in the innate immune system. BPI was initially isolated from rabbit granulocytes and serum, and then cloned and identified in various animals such as humans, cattle and rabbits. BPI has various functions such as antibacterial and bactericidal, neutralizing endotoxin, and immune regulation. The important advantage of BPI is that it is the azurophilic granule component of neutrophils, which is an antibacterial component of animals itself, and there is no problem of drug resistance, and at the same time, it does not cause any dama...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C12N15/70C12N1/21C07K14/435C07K1/36C07K1/34C07K1/30C07K1/22A61P31/04C12R1/19
CPCC07K14/43504C12N15/70A61P31/04
Inventor 张虹朱鹏曾达许尤厚龚斌宋静静方怀义邹起庭彭富强钟慧镁
Owner BEIBU GULF UNIV
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