问号赖型钩体内鞭毛基因重组质粒的构建及其在大肠杆菌的表达

Cloning, Characterization and Expression in E.coli of the Flagellin Gene from Leptospira interrogans Serovar lai

为了获得重组表达的钩体内鞭毛亚单位蛋白抗原，以探讨制备新疫苗的途径，利用聚合酶链反应扩增其编码基因完整的开放阅读框架，扩增产物经ＳａｌⅠ、ＣｌａⅠ双酶切消化后定向克隆于ｐＴ７－７表达载体上。重组质粒转化入大肠杆菌ＪＭ１０９（ＤＥ３）中，经ＩＰＴＧ诱导表达，ＳＤＳ－ＰＡＧＥ显示此产物相对分子量为３４ｋｄ，产量占细菌总蛋白的１１．８％。用插入片段为探针，Ｓｏｕｔｈｅｒｎ杂交提示可能有两个相关的基因存在于钩体的基因组内。Ｗｅｓｔｅｒｎ杂交证实该表达蛋白可与内鞭毛抗血清在３４ｋｄ处出现印迹，与钩体内鞭毛同类蛋白带位置一致，本实验克隆了钩体内鞭毛Ｂ类基因并表达了相应抗原。

To obtain a large amount of recombinant endoflagellin protein of leptospsira, we have cloned the fla B gene from Leptospira interrogans serovar lai strain 017, which encodes the core flagellar protein. The entire open reading frame was amplified by polymerase chain reaction, which was digested with endonucleases SalⅠ and ClaⅠ, then was orientationally cloned into the expression vector, pT7 7. The recombinant plasmid was transformed into Escherichia coli JM109 (DE3) for expression. After induction with IPTG, a 34kd expression protein was detected by SDS PAGE. The amount of recombinant protein was estimated as 11.8% of the whole bacterial lysate proteins. Probed with insert fragment, Southern blot analysis indicated that possibly two gene related to fla B were present in the genome of Leptospira interrogans serovar lai stain 017. Western blot analysis showed that the 34kd expression protein reacting with rabbit polyclonal antiserum raised against leptospira endflagellin.