关键词: 单核细胞增多性李斯特菌, iap基因, 克隆表达, p60蛋白, 纯化

Abstract:

In this study, iap gene of Listeria monocytogenes was amplified by PCR using self-designed primers and genomic DNA of Listeria monocytogengs as the template. Through introducing EcoRⅠand XhoⅠsites to the 5＇and 3＇ ends of iap gene, iap gene was inserted into pMD18-T vector and recombinant pMD-18T-iap plasmid was constructed. After the verification of DNA sequence, the iap gene was subcloned into expression vector pET-28a to obtain pET-28a-iap expression plasmid. Through ITPG induction of pET-28a-iap, the recombinant p60 protein with relatively molecular mass of 60 kD was over-expressed in E. coli BL21(DE3). The p60 protein was composed of soluble protein and inclusion protein and the amount of soluble protein reached up to 76.3%. The soluble protein was purified by Ni2+-chelating affinity column chromatography with an extraction rate of 68.3%, and the purity of recombinant protein was 95.6%. These results will provide a reference for further studies on structure and functions of p60 protein.

Key words: Listeria monocytogenes, iap gene, clone, expression, p60 protein, purification