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Probiotic clone strain for integrating four copy functional F18 fimbria operon genes, construction method and application

A construction method and operon technology, applied in the field of stably genetically expressing exogenous functional F18 fimbriae protein, can solve the problems of slow growth, weight loss, high mortality of piglets, etc., and achieve the purpose of reducing adhesion and improving adhesion performance. Effect

Active Publication Date: 2019-03-22
YANGZHOU UNIV
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Problems solved by technology

[0005] Piglet weaning diarrhea (PWD) and piglet edema disease (ED) are clinically common bacterial diseases in the pig industry. The main pathogens are F4 and F18 pili-positive enterotoxigenic Escherichia coli ETEC, which adhere and colonize susceptible piglets through pili Post-infection onset, leading to high piglet mortality, weight loss, slow growth, expensive drug treatment, etc.

Method used

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  • Probiotic clone strain for integrating four copy functional F18 fimbria operon genes, construction method and application
  • Probiotic clone strain for integrating four copy functional F18 fimbria operon genes, construction method and application
  • Probiotic clone strain for integrating four copy functional F18 fimbria operon genes, construction method and application

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Embodiment Construction

[0029] The present invention will be described in further detail below through specific examples.

[0030] The famous probiotic Escherichia coli Nissle 1917 (EcNc) is a non-pathogenic E. coli probiotic, which has been proven to give health benefits to different hosts, and E. coli Nissle1917 is a non-pathogenic strain that can give different hosts Health benefits, no known adverse effects on the host were found, and this strain is used as the main active ingredient of a probiotic product named Mutaflor in Europe, which is mainly used for the regulation of human intestinal health. The famous probiotic Escherichia coli Nissle 1917 (EcNc) was deposited in CGMCC, General Microorganism Center of China Committee for Culture Collection of Microbial Cultures, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the date of preservation was July 2018. 2. The deposit number is CGMCC No.16045.

[0031] In this ex...

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Abstract

The invention relates to the technical field of biology, and specifically relates to a probiotic clone strain for integrating four copy functional F18 fimbria operon genes, a construction method and application. The construction method comprises the following steps: constructing a pTargetT-X::PtetF18 recombinant plasmid; and constructing the probiotic clone strain for integrating four copy functional F18 fimbria operon genes to obtain a nonresistant four copy F18 fimbria gene integrated clone strain, and expressing and showing the clone strain on the surface of a cell. Compared with the priorart, the probiotic clone strain disclosed by the invention has the beneficial effects: original plasmid of Nissle 1917 probiotics is removed, and resistance genes are avoided; recombinant bacteria caneffectively improve adhesion property to a porcine intestinal tract passage epithelial cell (IPEC-1); by means of oral immunization, immune serum, namely anti-F18 fimbria IgG antibody, can be generated in a mouse; and after oral immunization, mouse serum can obviously reduce adhesion of a F18<+> bacterial strain to IPEC-J2, a porcine intestinal tract passage cell line.

Description

technical field [0001] The invention relates to the application field of biotechnology, in particular to the chromosomal integration of bacteria and the stable genetic expression of exogenous functional F18 pili protein. In the present invention, the CRISPR / cas9 dual-plasmid system has the characteristics of efficiently editing bacterial genomes (including integration insertion, knockout, and point mutation), and the CRISPR / cas9 dual-plasmid system has a mechanism for easy removal of its own plasmid and no residual resistance gene. Completed the construction of Escherichia coli probiotic Nissle1917 plasmid-free clone EcNc, which stably carries four copies of the F18 pili operon gene on the surface and genetically expresses F18 pili, without affecting the biological characteristics of the host strain EcNc. Anti-F18 + A new method of probiotic live vaccine candidate strains for post-weaning diarrhea or edema disease in piglets caused by the strains, enabling the preparation of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/90C12N15/31A61K39/108A61P31/04C12R1/19
CPCA61K39/0258A61P31/04C12N15/902C07K14/245A61K2039/552A61K2039/522
Inventor 朱国强区炳明金铎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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