E型产气荚膜梭菌ι毒素重组质粒的构建与表达

Construction and Expression of Iota Toxin Recombinant Plasmid of Clostridium perfringens Type E

ι毒素是是公认的引起羔羊、犊牛和家兔患肠毒血症的主要毒素,为了减小其给畜牧业带来重大损失,论文就ι毒素蛋白的构建与表达进行研究,以期对其所引起疾病的预防及疫苗的研发提供参考。通过E型产气荚膜梭菌基因组,利用PCR方法获得大小为1 377bp的iota a基因和2 640bp的iota b基因;随后将目的基因同pET-28a质粒连接构建重组载体,并将重组载体转化E.coli BL21,用IPTG诱导表达,并通过SDS-PAGE、Western blot进行分析,发现Ia、Ib蛋白均成功进行了可溶性表达;最后利用Ni-NTA层析柱对重组蛋白进行纯化,获得了目的蛋白。

Being known as the major cause of enterotoxaemia, Iota-toxin has caused great losses to animal husbandry yearly. To bring down the loss and to figure out the prevention and the vaccine of such disease, the expression and purification of Iota-toxin was recorded in this study. Clostridium perfringens type E genome was used as template for the PCR. The genetic engineering methods were used to construct the recombinant vector,the iota a gene（1 377 bp） and iota b gene（2 640 bp） were amplified from C. perfringens type E genomc DNA by PCR. The genes were cloned into pET-28a vector and expressed in E. coli BL21. The SDS-PAGE and Western blot analyses showed that the recombinant proteins were mainly in the form of soluble expressiors. The results indicated that the recombinant proteins possessed strong antigenicity which could be used as gene engineering vaccine and small molecular antibody.