Seyed Sayyah P, Golestani B, Pilehchian Langroud R
Department of Biology, Islamic Azad University, Urmia branch, Urmia, Iran.
Specialized Clostridia reseach laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Alborz, Karaj, Iran.
Arch Razi Inst. 2018 Jun;73(2):107-111. doi: 10.22092/ari.2018.116618. Epub 2018 Mar 1.
Iota toxin is produced by Clostridium perfringens type E. This toxin causes antibiotic-associated enterotoxemia in lambs and calves. Iota toxin is a binary toxin that has two components including Ia (the enzyme component) and Ib (the binding component). Ib binds to the surface receptor of target cells and translocate Ia into the cytosol of cells. The aim of this study was to clone toxigenic epitope of iota a gene in E. coli strain Top10. In this study, the phenol–chloroform method was used for the extraction of the whole genomic DNA. The toxigenic epitope of iota a gene was amplified by polymerase chain reaction (PCR). The PCR product was ligated into the pTZ57R/T vector cloning site. Then, based on the TA-cloning method, the product was cloned in competent E. coli strain Top10. Colony PCR was used to screen bacterial colonies transformed with recombinant plasmids. The presence of 446-bp fragment on agarose gel showed that the toxigenic epitope of iota a gene of C. perfringens has been cloned in E. coli strain Top10.
埃塔毒素由产气荚膜梭菌E型产生。这种毒素会导致羔羊和犊牛出现抗生素相关性肠毒血症。埃塔毒素是一种二元毒素，有两个组分，包括Ia（酶组分）和Ib（结合组分）。Ib与靶细胞的表面受体结合，并将Ia转运到细胞胞质溶胶中。本研究的目的是在大肠杆菌Top10菌株中克隆埃塔a基因的产毒表位。在本研究中，采用酚-氯仿法提取全基因组DNA。通过聚合酶链反应（PCR）扩增埃塔a基因的产毒表位。将PCR产物连接到pTZ57R/T载体克隆位点。然后，基于TA克隆法，将产物克隆到感受态大肠杆菌Top10菌株中。用菌落PCR筛选用重组质粒转化的细菌菌落。琼脂糖凝胶上446 bp片段的出现表明产气荚膜梭菌埃塔a基因的产毒表位已在大肠杆菌Top10菌株中克隆。
