李斯特细菌hlyA的原核表达与纯化  

Prokaryotic expression,purification of Listeria monocytogenes hlyA

作　　者：韩乐[1] 高志贤[1] 宁保安[1] 孙志勇[1] 

机构地区：[1]中国军事医学科学院卫生学环境医学研究所,天津300050

出　　处：《哈尔滨医科大学学报》2014年第2期124-127,共4页Journal of Harbin Medical University

摘　　要：目的构建李斯特细菌毒力基因原核表达质粒,并诱导其表达纯化并鉴定目的蛋白。方法构建李斯特溶血素O(Listeriolysin O,LLO)的原核表达载体PGEX/3X,并利用Factor Xa切割了GST标签,酶切正确后构建重组原核表达质粒,并转化到大肠杆菌,IPTG诱导目的蛋白表达并进行Western blot鉴定。结果目的基因经酶切其结果与预期相符,测序结果显示目的基因GenBank登陆的序列完全一致,重组质粒经IPTG诱导表达相对分子量为60 kD的目的蛋白;Western blot鉴定结果显示,重组蛋白能够与抗LM抗体特异性结合。结论成功构建了重组原核表达质粒,并纯化获得高纯度的LLO蛋白。Objective To clone the hlyA gene, construct prokaryotic plasmid of hlyA and express it in prokaryotic plasmid, and purify and identify the target protein. Methods LLO protein prokaryotic expression vector PGEX/3X was constructed, and the GST tag was cut by using Factor Xa, and the recombinant prokaryotic expression vector was constructed, and was transformed into E. coli. IPTG induction of protein expression and was identified by Western blot. Results The identification of target gene by restriction enzyme was the same as the expectation. The sequence of target gene was identical with that registered in GenBank. With induction of IPTG, the recombinant plasmid expressed a new fusion protein with relative molecular mass of 60 kD; The identification results of Western blot showed that the recombinant protein can be specific binding with anti-LLO antibody. Conclusion The recombinant prokaryotic expression plasmid is constructed successfully, and the recombinant LLO protein with highly purity concentration is gained.

关 键 词：李斯特菌 hlyA 原核表达纯化 

分 类 号：R378.99[医药卫生—病原生物学]