Integrated double copy functional F4 pili operon gene porcine probiotic EP1 clone strain, construction method and application

A construction method and double-copy technology, applied in the field of biotechnology applications, can solve problems such as high mortality, weight loss, and slow growth of piglets, and achieve the effect of reducing the number of adhesions and improving adhesion performance

Active Publication Date: 2019-04-16
YANGZHOU UNIV
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Problems solved by technology

[0005] Piglet post-weaning diarrhea (PWD) is a common clinical disease in the pig industry. The main pathogen is F4 pili-positive enterotoxigenic Escherichia coli ETEC,

Method used

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  • Integrated double copy functional F4 pili operon gene porcine probiotic EP1 clone strain, construction method and application
  • Integrated double copy functional F4 pili operon gene porcine probiotic EP1 clone strain, construction method and application
  • Integrated double copy functional F4 pili operon gene porcine probiotic EP1 clone strain, construction method and application

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Embodiment Construction

[0027] The present invention will be described in further detail below through specific examples.

[0028] NY10 (EP1) is a non-pathogenic E. coli probiotic isolated and preserved in our laboratory, and it has been verified by whole genome sequencing. E. coli NY10 (EP1) is a non-pathogenic strain that can give pigs intestinal Health benefits, no known adverse effects on pigs were found.

[0029] In this experiment, probiotic NY10 (EP1) was used as a vaccine carrier to construct recombinant probiotics stably expressing F4 pili, which were used as daily feed additives to immunize piglets to prevent and control post-weaning diarrhea (PWD) in piglets. The F4 pilus-integrated probiotics can specifically bind to the F4 pilus receptor of porcine intestinal cells, preferentially occupy the receptor site, and induce mucosal immunity against F4 E. coli pathogens, thereby blocking F4 + Adhesion of ETEC to piglet intestines is aimed at highly effective and direct prevention of post-weanin...

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Abstract

The invention relates to thefield of biotechnology, in particular to an integrated double copy functional F4 pili operon gene porcine probiotic EP1 clone strain, a construction method and application.The method comprises the following steps that pTargetT-maeB::PtetF4 and pTargetT-nth/tppB::PtetF4recombinant plasmids are constructed; a non-resistant doublecopy F4 pili geneintegrated clone strain is obtained by constructing the probiotic clone strain of an integrated doublecopy functional F4 pili operon gene. The probiotic clone strain is used as a probiotic live vaccine candidate strain and atherapeutic drug for post-weaning diarrhea and an edema disease in piglets. Compared with the prior art, the strain has the advantages that a porcine probiotic NY10 (EP1) and a non-resistant gene exist, recombinant bacteria can effectively improve the adhesion performance to intestinal passage cells of pigs, cell infection inhibition experiments in vitroshow that pre-incubationrecombinant bacteriaand IPEC-J2 cells can competitively inhibit the infection of F4 piluspositive E. coli pathogen 3030-2 against the IPEC-J2 cells, and the number of adhesion is significantly reduced.

Description

technical field [0001] The invention relates to the application field of biotechnology, in particular to the stable genetic expression of exogenous functional F4 pilus protein in Escherichia coli. Background technique [0002] Escherichia coli NY10 (EP1) was isolated from the feces of non-diarrhea pigs in pig farms with severe diarrhea in the 1950s in our laboratory. It has a good preventive effect on pig diarrhea. It is a pig-derived E. coli probiotic. The Escherichia coli probiotic has a beneficial effect on the intestines of pigs, and no known adverse effects on the host have been found so far, and the applicant's laboratory has analyzed and verified its beneficial effect through whole genome sequencing of the pig-derived Escherichia coli probiotic. genetic background of bacteria. [0003] In order to achieve the overexpression of homologous or heterologous proteins or compounds in microorganisms, overexpression of plasmids is mostly used. This method is easy to operate ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/90C12N15/31A61K35/741A61P1/12A61P7/10C12R1/19
CPCA61P1/12A61P7/10C12N15/902C07K14/245A61K2035/115Y02A50/30
Inventor 朱国强金铎区炳明夏芃芃朱军徐梦娴宋浩亮梁轩杨颖朱晓芳
Owner YANGZHOU UNIV
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