产肠毒素性大肠杆菌致病因子F_(41)、K_(99)菌毛基因的克隆与表达

Clone and expression of pili subunit gene from the pathogenic factor adhesion F41 and K99 of the Enterotoxigenic Escherichia coli(ETEC)

目的为了分析原核表达的ETECF41和K99菌毛亚单位的抗原性和作为基因工程亚单位疫苗的可能性。方法针对黏附素F41和K99的编码序列,设计两对特异性引物,扩增F41和K99菌毛蛋白的编码基因,经序列测定后,克隆到原核表达载体pET-28a(+)上,转化E.coliBL21(DE3),筛选到可以表达F41和K99菌毛亚单位的菌株。经IPTG诱导表达后,进行SDS-PAGE和Western blot试验分析原核表达的F41和K99重组蛋白的抗原性。结果表达的F41和K99菌毛亚单位蛋白可以与抗F41和K99菌毛蛋白的单因子血清反应。结论原核表达的F41和K99重组蛋白具有良好的抗原性。

In order to analyze the antigenicity of pili subunit of ETEC adhesin F41 and K99 and the possibility to use them as candidate in the subunit vaccine,two pairs of primers were designed according to the coding sequence of these adhesin genes and used for the amplification of the coding gene for these pili proteins, The full-length of the F41 and K99 fragments were cloned into the prolaryotie expression veetor pET-28a and the expression of target protein in E. coli BL21 was determined by SDS-PAGE and Western blotting. It was found that the expressed F41 and K99 pili subunit proteins could react with antiserum against the single factor of F41 or K99 pili subunit proteins,thus proving the antigenicity of these proteins.