牛微小隐孢子虫子孢子表面抗原p23基因的克隆及原核表达

MOLECULAR CLONING AND PROKARYTIC EXPRESSION OF Cryptosporidium parvumSURFACE ANTIGEN P23 GENE IN Escherichia coliBL21

从已构建的牛微小隐孢子虫子孢子cDNA文库中,用PCR方法扩增出子孢子表面抗原p23基因,将其插入到编码谷胱肽转移酶(GST)的质粒(pGEX-4T-2)多克隆位点,构建原核表达载体pGEX-p23。重组质粒经EcoRI/Sal I酶切、测序鉴定后转入大肠杆菌BL21中,IPTG诱导表达目的蛋白,SDS-PAGE和Western blot鉴定表达蛋白。结果成功构建了pGEX-p23原核表达载体,经IPTG诱导,转化的大肠杆菌表达出分子质量为47kDa的GST融合蛋白,且该蛋白能与抗p23抗体发生特异性结合反应。本研究为进一步研究p23的生物学功能和临床应用奠定了基础。

Total DNA was extracted from cDNA library of Cryptosporidium parvum and the DNA fragment encoding mature peptide of p23 gene was amplified by PCR method. The fragment was cloned into plasmid pGEX -4T -2, forming the recombinant prokaryotic expression vector （pGEX -p23） which＇was identified by EcoR I and Sal I digestion and sequence analysis. The E. coli BI21 contai- ning recombinant plasmid was induced by IPTG to express fusion protein. SDS - PAGE and Wastem blot were performed to identify the recombinant protein. SDS -PAGE analysis showed that there was a new protein band, which is of 47kDa. Western blot proved that the fusion protein could be specifically recognized by anti - p23 antibody and it is the basis for the study on biological activities of P＇23 and for possible clinic application in the future.