大肠杆菌F18ac菌毛FedA蛋白的表达与鉴定

Expression and Identification of the Major Subunit FedA of the Fimbriae F18ac of Escherichia coli

根据已经发表的F18ac菌毛A亚单位的基因序列(FedA)设计一对引物,利用PCR技术从重组质粒T 8813A 中扩增到一段序列,并按预定的阅读框插入表达性质粒载体pGEX 6p 1中的谷胱苷肽转移酶(GST)基因的下游,获得重组质粒pPFedA/ac,并转化大肠杆菌BL 21 获得重组菌PPFedA/ac。琼脂糖凝胶电泳、序列测定及分析表明,该序列大小为456 bp,与已发表的FedA/ac结构编码序列完全一致。通过对菌体裂解物的SDS PAGE 分析以及Western blotting 鉴定,证明重组大肠杆菌PP FedA/ac的可以表达融合蛋白形式的FedA/ac (命名为GST FedA/ac),即FedA/ac蛋白(15.317 ku)与谷胱苷肽转移酶(27.335 ku)相连组成分子量为42.652 ku的融合蛋白。利用GST FedA/ac制备的兔抗GST FedA/ac 血清与大肠杆菌F107/86 株( F18ab+ )、2 134 P 株(F18ac+)进行的玻板凝集试验呈现阳性反应,进一步表明了表达的正确性。

A pair of primers were designed and synthesized according to the major subunit FedA gene of fimbriae F18ac, and the subgenic fragment of fedA/ac was amplificated from the recombined plasmid T8813A, with appropriate restriction sites. The plasmid vector pGEX-6p-1 was used for the expression ofthe fedA/ac. The 5' terminus of the gene that code for fedA/ac was genetically fused to the 3' terminus of the gene coding for the enzyme glutathione S-transferase, which serves as a carrier in this expression system. The resulting plasmid contained the fedA/ac E gene was designed as pPfedA/ac. The pP fedA/ac was transferred into E.coli BL21 and confirmed that it was able to express large quantities of a 42.652 ku fusion protein (GST-FedA/ac) by SDS-PAGE analysis and Western blotting. The data that rabbit serum against GST-FedA/ac can reacted with fimbriae F18 positive strains, such as F107/86 and 8813, were farther confirmed by successful expression of FedA/ac.