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本研究利用λRed重组系统构建了肠毒性大肠杆菌(ETEC)H10407菌的gsp D基因敲除株,并利用PCR和测序技术进行了验证。随后对gsp D基因敲除株和野生株生长曲线进行测定,并利用Western Blot技术对培养上清液和细胞沉淀中的LT毒素分别进行检测。发现gsp D基因敲除对ETEC H10407生长无显著影响,野生型大肠杆菌的LT毒素不仅存在于培养上清液中,还存在于细胞沉淀,而gsp D基因敲除株的LT毒素仅在细胞沉淀中被检测到。本研究的结果表明gsp D基因敲除的ETEC,其LT毒素在细菌细胞内积累并不释放至细胞外,说明Ⅱ型分泌系统(T2SS)中的gsp D基因对调控LT毒素分泌至细胞外起到了关键作用。
In this study, we constructed the gsp D knockout strain of enterotoxigenic Escherichia coli (ETEC) H10407 using lambda Red recombination system and verified by PCR and sequencing. Subsequently, the growth curves of gsp D knockout and wild strains were determined, and the LT toxins in the culture supernatant and cell pellet were detected by Western Blot. It was found that gsp D knockout had no significant effect on the growth of ETEC H10407. The LT toxin of wild-type Escherichia coli was not only present in the culture supernatant but also in the cell pellet, whereas the LT toxin of the gsp D knockout strain was only expressed in the cell pellet Was detected. The results of this study showed that ET-1 knockout in gsp D knock-out ETECs accumulated in bacterial cells and not released to the outside of cells, indicating that the gsp D gene in type II secretion system (T2SS) regulates the secretion of LT toxin To the key role.