变形假单胞菌ExsA蛋白表达、纯化及多克隆抗体的制备

doi:10.3969/j.issn.1674-7968.2023.04.020

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Abstract TypeⅢsecretion system (T3SS) is ubiquitous transport machinery in Gram-negative bacteria and an important way for bacterial virulence factors to produce pathogenic effects. Ara-C type DNA binding protein ExsA is one of the main activators of T3SS and can regulate the expression of T3SS-related proteins. In this study, ExsA gene of Pseudomonas plecoglossicida PQLYC4 strain was cloned into the prokaryotic expression vector pET28a (+). The recombinant expression plasmid pET-28a-ExsA was transformed into competent cells of Escherichia coli BL21 (DE3) for induced expression. The results showed that the molecular mass of the expressed recombinant ExsA protein was slightly less than 30 kD, which was consistent with the expected molecular mass (29.7 kD). The purified recombinant ExsA protein was used as an antigen to immunize rabbits (Oryctolagus cuniculus) to prepare polyclonal antibodies, and the titer was above 1∶256 000. The prepared antibody were used to detect ExsA protein in P. plecoglossicida and the over expressed ExsA protein in the epithelioma papulosum cyprini (EPC) of carp (Cyprinus carpio) by Western blot and indirect immunofluorescence methods, the results showed specific reactions. This study provides a tool for further exploring the structure of ExsA protein and its function in regulating the virulence factor T3SS system of P. plecoglossicida.

Key words： Pseudomonas plecoglossicida ExsA Prokaryotic expression Protein purification Polyclonal antibody

Received: 13 June 2022

ZTFLH:

S917.1

Corresponding Authors: * panqin@fafu.edu.cn

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	Articles by authors
	WEI Jin-Ping
	LI Yun-Feng
	QIU Wei
	CHEN Xing-Pu
	CHEN Bo-Yu
	CHEN Xin-Hua
	QIN Pan

Cite this article:

WEI Jin-Ping,LI Yun-Feng,QIU Wei, et al. Expression, Purification and Polyclonal Antibody Preparation of Pseudomonas plecoglossicida ExsA Protein[J]. 农业生物技术学报, 2023, 31(4): 883-888.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2023.04.020 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2023/V31/I4/883

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