猪水肿病大肠杆菌F18ab菌毛A亚单位与志贺样毒素Ⅱ型变异体A亚单位的联合表达

Coexpression of a Subunit of Fimbriae F18ab and a Subunit of Shiga-Like Toxin Type Ⅱ Variant(Stx 2eA)

扩增、克隆和表达了水肿病大肠杆菌的致病因子F18ab 菌毛的主要亚单位FedA全基因(包括信号肽序列),并与现有的另一致病因子志贺样毒素Ⅱ型变异体A亚单位基因(stx 2eA)连接,然后进行联合表达。同时用已制备的抗Stx 2eA 单克隆抗体和抗F18ab菌毛单克隆抗体对联合表达的融合蛋白质进行检测鉴定。

Edema disease (ED) of weaning piglets is caused mainly by Verotoxigenic Escherichia coli (VTEC) which lead to edema in various tissues including the central nervous system. Fimbriae F18ab and variant of the Stx 2e toxin were two pathogenic factors of VTEC.The A subunit gene (FedA, 510 bp) of the F18ab fimbriae was amplificicated from Escherichia coli 107/86 by polymersae chain reaction (PCR).The PCR product of FedA was cloned into plasimid vector pGEX-6P-1 between Bam H Ⅰ and EcoRⅠ sites.The gene was genetically inserted at the downsteam of the 3`terminus of the gene coding for enzyme glutathione S-transferase, which served as a carrier in this expression system.The coding sequence of Stx 2eA(stx 2eA, 900 bp) was amplified from pSLT-IIeA and was cloned into pF107 at Eco R Ⅰ and Sal I sites, the resulting recombinants was called pFStx2eA.The result of SDS-PAGE and Western-blot assay demonstrated that the fusion protein(GST-F18ab-Stx 2eA) expressed by the recombinant plasmid pFStx2eA can specifically response to the monoantibody against F18ab fimbriae and Stx 2eA.This indicated that the expected co-expression protein was successfully obtained.