牛源产Ⅱ型不耐热肠毒素大肠杆菌的分离鉴定及重组毒素制备

Isolation of Type Ⅱ Heat-labile Enterotoxin-producing Escherichia coli and Preparation of the Recombinant Toxin

产Ⅱ型不耐热肠毒素(LT-Ⅱ)大肠杆菌是一类能引起严重腹泻的人畜共患肠道致病菌。为了解LT-Ⅱ在牛源腹泻性大肠杆菌中的流行情况,本研究对2010-2012年间分离的138株大肠杆菌进行了LT-Ⅱ毒素基因的检测,对分离到的10株LT-Ⅱ阳性大肠杆菌进行了测序及分析。进一步将LT-Ⅱcl毒素基因双顺反子的ORF克隆入pET30a表达载体、转化Rossatta感受态并诱导表达重组LT-Ⅱcl毒素,经Y-1小鼠肾上腺皮质细胞对重组毒素进行了初步的毒性验证。结果显示,所分离的10株LT-Ⅱ阳性大肠杆菌中,有8株表达LT-Ⅱcl毒素,1株表达LT-Ⅱc4毒素,1株表达LT-Ⅱc6毒素。SDS-PAGE和Western Blot结果表明成功地制备出LT-Ⅱcl重组毒素,该重组毒素可以使Y-1小鼠肾上腺皮质细胞发生明显的病变,其细胞最小致死量为17.8 pg。本研究为中国国内首次分离到牛源产LT-Ⅱ型不耐热肠毒素大肠杆菌,为深入研究LT-Ⅱ毒素奠定了基础。

Type Ⅱ heat-labile enterotoxin(LT-Ⅱ)-producing Escherichia coli is a common zoonotic pathogenic agent responsible for diarrhea.In order to improve the understanding of the epidemiological situation of LT-Ⅱ toxin in cattle,a total of 138 E.coli isolates from 2010 to 2012 were detected for LT-Ⅱ gene by PCR,and ten of these were sequenced.The polycistron ORF of LT-Ⅱcl was cloned into the pET30 a expression vector,translated into Rossatta competent cells,and induced for expressing the recombinant LT-Ⅱ toxin.The toxicity of the recombinant LT- Ⅱtoxin was preliminary tested by mouse Y1 adrenal cells.The results showed that eight samples were LT-Ⅱ cl producing E.coli,one was LT-Ⅱ c4 producing E.coli and one was LT-Ⅱc6 producing E.coli from ten LT-Ⅱ positive.The results of SDS-PAGE and Western Blot showed that recombinant LT-Ⅱ cl toxin was prepared successfully,and the recombinant toxin had induced the apparent cytopathic effect for mouse Yl adrenal cells,with the minimum lethal dose of 17.8 pg.The results presented here demonstrate that we isolated the LT-Ⅱ producing E.coli for the first time in China,and lay a preliminary foundation for further study of the LT-Ⅱ toxin.