【摘要】 根据已发表的F18菌毛F亚单位的基因序列(fedF)设计了1对引物,利用PCR技术分别扩增到F18ab和F18ac菌毛的fedF编码序列,并按预定的阅读框架分别插入表达载体pGEX 6p 1中谷胱甘肽转移酶(GST)基因的下游,获得重组质粒pPFedF/ab与pPFedF/ac,然后转化大肠埃希氏菌BL21获得重组菌PPFedF/ab与PPFedF/ac。测序结果表明,fedF编码序列大小均为837 bp,与GenBank中登录的FedF 结构编码序列(Z26520)大小一致。通过对菌体裂解物进行SDS PAGE电泳分析以及Western blotting鉴定,证明重组大肠埃希氏菌PPFedF/ab与PPFedF/ac均可以表达融合蛋白形式的FedF(分别命名为GST FedF/ab与GST FedF/ac)。更多还原

【Abstract】 A pair of primers was designed and synthesized accordingto the minorsubunitFedF genes of fimbriae F18ab and F18ac, and the subgenic fragmentsoffedF/ab and fedF/acwere amplified from the recombined plasmids TF107F and T8813F respectively. The plasmid vector pGEX-6p-1 was used for the expression of the fedF/ab and fedF/ac. The 5′ terminal of the genes encoding the interestedproteinsweregenetically fused to the 3′ terminal of the gene encoding the enzyme glutathione S-transferase, which was used as a carrier in this expression system. The plasmids containing the fedF/ab or fedF/ac gene were named aspPFedF/ab or pPFedF/ac respectively. The interested recombined plasmids pPFedF/ab and pPFedF/acwere transferred into E.coli BL21 respectively. It was confirmed by SDS-PAGE analysis and Western-blotting that they were able to express large quantities of the 57.539ku fusion protein(GST-FedF/ab and GST-FedF/ac).更多还原