中国钩端螺旋体强毒株017膜蛋白基因的质粒载体构建及表达

目的构建表达我国钩体强毒赖型０１７株外膜蛋白的重组质粒。方法用ＰＣＲ方法扩增并回收０１７株钩体外膜蛋白基因片段ＯｍｐＬ１，将其与质粒ｐＢＫ－ＣＭＶ重组，通过菌落颜色和酶谱分析筛选出重组质粒。然后从该重组质粒切下ＯｍｐＬ１基因片段，重新克隆至质粒ｐＢＶ２２０中，酶谱分析和ＤＮＡ杂交鉴定出正向重组质粒。将含有正向重组质粒的宿主菌诱导表达生长后，提取全菌总蛋白进行ＳＤＳ－ＰＡＧＥ分析。结果筛选出４个与ｐＢＶ２２０正向重组的质粒，其中一株带重组质粒ｐＢＬＭ１的菌株表达了一相对分子质量为３３．５×１０３的新蛋白。结论重组质粒ｐＢＬＭ１的构建和表达成功说明扩增得的ＯｍｐＬ１基因具有完整的阅读框架，并使简便获得大量天然的０１７株钩体ＯｍｐＬ１蛋白成为可能，为新型钩体疫苗的研究奠定了基础

Construction and expression of the plasmid vector of an outer envelope protein gene from the strong virulent Leptospira,strain 017 in China

Objective Constuction of a recombinant plasmid expressing an outer envelope protein from the strong virulent Leptospira , serovar Lai strain 017. Methods The OmpL1 outer envelope protein gene was amplified from the genome of the Leptospira , strain 017. The removed OmpL1 gene was recombined with plasmid pBK CMV and the recombinant plasmid was selected according to the colour of bacterial colony and analysis of restriction enzymes. Then the OmpL1 gene was cut out from the recombinant plasmid and was cloned to plasmid pBV220 again. The recombinant plasmids with proper orientation was identified with analysis of restriction enzymes and DNA hybridization and selected . The host bacteria containing recombinant plasmids with proper orientation grew with heat induction , and the complete protein of the bacteria was extracted for SDS PAGE. Results Four recombinant plasmids with proper orientation, which ligated with pBV220, were obtained. One of the four recombinant plasmids, pBLM1, expressed a new protein with a weight of 33.5kD. Conclusions The success of construction and expression of recombinant plasmid pBLM1 has indicated that the OmpL1 gene obtained by PCR has a complete reading frame. And it supposes to be possible to use strain 017 for getting natural OmpL1 protein of Leptospira in quantity. The above work has laid a necessary foundation for future research on a new vaccine of Leptospira .