Application of flavobacterium columnare virulence protein in pathogen detection and low virulent strain preparation

A technology of Flavobacterium columnar and attenuated strains, applied in the field of molecular biology, can solve the problems of low homologous recombination efficiency, difficulty in exogenous genes, and difficulty in genetic modification, etc.

Active Publication Date: 2022-03-08
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Flavobacterium columnar belongs to the Bacteroides phylum. Compared with the efficient genetic manipulation in the Proteobacteria phylum, many pathogenic bacteria of the Bacteroidetes phylum are difficult to introduce foreign genes, and the efficiency of homologous recombination is low, making genetic transformation difficult.
Therefore, the researchers also speculated that some possible virulence factors of Flavobacterium columnar, such as: chondroitin lyase, collagenase and outer membrane protein A (OmpA), but did not find their relationship with Direct evidence for virulence, only its expression level was examined (Laanto et al., 2014; Penttine et al., 2016)

Method used

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  • Application of flavobacterium columnare virulence protein in pathogen detection and low virulent strain preparation
  • Application of flavobacterium columnare virulence protein in pathogen detection and low virulent strain preparation
  • Application of flavobacterium columnare virulence protein in pathogen detection and low virulent strain preparation

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Experimental program
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Effect test

Embodiment 1

[0038] Obtaining the virulence protein of Flavobacterium columnar:

[0039] According to the whole genome sequences of 62 strains of Flavobacterium columnar that have been published so far, combined with the infection characteristics of Flavobacterium columnarum, the possible virulence genes were screened out. The exogenously expressed proteins of these genes are obtained through in vitro expression and affinity purification. These proteins were co-incubated with fish cells to detect their virulence. It was found that only protein cytolysin with Thiol-activated cytolysin superfamily domain could cause fish cell pathology. After comparing the two cytolysin coding sequences ctl1 and ctl2, it was found that although the two sequences are highly similar and have the same structural domain (Thiol-activated cytolysin superfamily), only the prokaryotic expression protein of ctl2 has a significant lytic effect on cells, so The amino acid sequence of the ctl1 is shown in SEQ ID NO.1,...

Embodiment 2

[0041] The virulence detection of prokaryotic expression proteins of Flavobacterium columnar ctl1 and ctl2 genes, the specific methods include:

[0042] (1) Prokaryotic expression and purification of Ctl1 and Ctl2 proteins

[0043] The 25-558aa region of the Ctl1 protein and the 23-558aa region of the Ctl2 protein were respectively cloned into the pGEX-4T-AB1 vector, and the expression plasmids were transformed into Escherichia coli Rosseta (DE3) strain. The strain carrying the expression plasmid was induced to express at 16°C for 16 hours, and subjected to crushing treatment. The crushed supernatant was purified and digested, the GST tag was removed, and the endotoxin was removed for later use.

[0044] (2) Preparation of polyclonal antibody against virulence protein cytolysin

[0045] Select the virulence protein cytolysin epitope synthetic polypeptide (KNLKQESIHDGPKSE) to prepare rabbit polyclonal antibody. The polyclonal antibody of Flavobacterium columnar cytolysin is ...

Embodiment 3

[0054] The construction of attenuated strain of Flavobacterium columnar:

[0055] (1) The initiation codon encoded by the open reading frame of the ctl1 gene and its upstream sequence (2084bp in total) are cloned into the PstI and SalI restriction sites of the Flavobacterium columnar suicide plasmid pMS75, and then the termination codon of the gene ORF The progeny and its downstream sequences (total 1989bp) were cloned into the BamHI and SalI restriction sites of the suicide plasmid pMS75. Finally, the suicide plasmid pNL-73 for ctl1 gene deletion was formed.

[0056] (2) Using the same method, clone the start codon of the ctl21 gene and its upstream sequence (2069bp in total) to the BamHI and SalI restriction sites of the suicide plasmid pMS75, and then clone the stop codon of the gene ORF and its downstream The sequence (2011bp in total) was cloned into the SalI and PstI restriction sites of the suicide plasmid pMS75. Finally, the suicide plasmid pNL-65 for ctl2 gene delet...

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Abstract

The invention belongs to the field of molecular biology, and discloses an application of a flavobacterium columnar virulence protein in pathogen detection and low virulent strain preparation, and the amino acid sequence of the flavobacterium columnar virulence protein is shown as SEQ ID NO.1 or SEQ ID NO.3. The invention further discloses a preparation method of the flavobacterium columnar virulence protein. An antibody is designed by utilizing the virulence protein and can be used for detecting pathogenic bacteria of flavobacterium columnar. A virulence gene for coding the protein is deleted from flavobacterium cylindricum, so that a flavobacterium cylindricum attenuated strain can be obtained, and the capability of the flavobacterium cylindricum for infecting a fish cell line and a fish body can be reduced by utilizing the attenuated strain; further, the strain is used as a candidate vaccine strain to realize immune prevention and control of columnar diseases caused by flavobacterium cylindricum.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to the application of a virulence protein of Flavobacterium columnar in pathogen detection and attenuated strain preparation. Background technique [0002] Flavobacterium columnare is the pathogen of fish columnar disease (also known as bacterial gill rot in my country). The bacterium is a Gram-negative bacterium that widely exists in the natural environment and artificial breeding environment around the world, and poses a serious threat to the freshwater aquaculture industry. Almost all economic fish species cultured artificially in my country, such as grass carp (Ctenopharyngodon idellus), bighead carp (Hypophthalmichthys nobilis), mandarin fish (Sinipercachuatsi), channel catfish (Ictalurus punctatus) and yellow catfish (Pelteobagrus fulvidraco), are highly susceptible to infection with this fungus. And cause huge economic losses (Administrative Administration of Fish...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C07K16/12C12N1/21C12N15/70A61K39/02A61P31/04C12R1/20C12R1/19
CPCC07K14/195C07K16/1203C12N15/70A61K39/02A61P31/04
Inventor 聂品李楠杨柳龙苏陈善楠李丽
Owner INST OF AQUATIC LIFE ACAD SINICA
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