单核增生性李氏杆菌溶血素的原核表达及可溶性分析

Prokaryotic Expression of Listeriolysin O(LLO) Gene in Escherichia coli and Analysis of Soluble LLO Protein

对单核增生性李氏杆菌的主要毒力基因hlyA进行大肠杆菌原核表达,分析其表达蛋白的可溶性并纯化。根据hlyA基因设计特异性引物,细菌煮沸破裂提取基因组,PCR扩增出目的条带。连接转化至克隆载体pMD18-T,测序正确后连接表达载体pET-30a(+),酶切和PCR鉴定正确后转化至BL21(DE3),构建重组质粒pET-30a-hlyA。IPTG诱导表达蛋白,并分析其可溶性。PCR扩增的hlyA基因全长约1 600 bp,成功构建了重组质粒pET-30a-hlyA,转化大肠杆菌诱导表达了溶血素蛋白,SDS-PAGE分析表达产物,结果显示表达的蛋白分子量约为60 kD,以可溶性和包涵体2种形式存在,且以可溶性为主要形式。成功克隆和表达了单核细胞增生性李氏杆菌hlyA基因和溶血素蛋白,并得到纯化蛋白。

The hlyA genes were the major virulence factor secreted by the intracellular pathogen Listeria monocytogenes.This research has expressed the hlyA gene by escherichia coli system.Specific primers were designed based on the hlyA gene and the target gene was amplified by PCR using its genome through boiling bacteria.HlyA gene was inserted into the cloning vectors pMD18-T.After the verification of DNA sequence,the hlyA gene was subcloned into expression vector pET-30a and recombinant plasmid pET-30a-hly was constructed.The hlyA gene amplified by PCR was 1 600 bp and the recombinant plasmid pET-30a-hlyA was successfully constructed.E.Coli induction expressed the LLO,SDS-PAGE was used to analyze products.The results show that the molecular weight of the expressed protein was 60 kD.It existed in the soluble form and in the form of inclusion body and the former was predominant.Conclusion： Listeria monocytogenes of hlyA genes and Listeriolysin O protein were successfully cloned and expressed,and purified protein was obtained.