问号钩端螺旋体鞭毛相关基因flhA、flhB_2和fliR的克隆及原核表达系统的构建

Cloning of Flagellar Biosynthesis Genes flhA,flhB_2 and fliR of Leptospira interrogans and Construction of Prokaryotic Expression System

目的克隆问号钩端螺旋体鞭毛相关基因flhA、flhB2和fliR,并构建其原核表达载体。方法按常规苯酚-氯仿法提取问号钩端螺旋体黄疸出血群赖型56601株基因组DNA,高保真PCR扩增flhA、flhB2和fliR基因片段,T-A克隆后测序,构建原核表达载体,采用SDS-PAGE和免疫印迹法检测目的融合蛋白的表达。结果问号钩体56601株flhA、flhB2和fliR基因扩增片段的核苷酸序列与报道的相应序列同源性分别为100%、99·9%和99·9%,氨基酸序列同源性分别为100%、99·8%和100%。所构建的重组原核表达系统在IPTG的诱导下,能有效地表达目的融合蛋白Trx-FlhA、Trx-FlhB2和Trx-FliR,产量约为细菌总蛋白的10%。结论已成功构建了问号钩端螺旋体鞭毛相关基因flhA、flhB2和fliR原核表达系统。

Objective To clone the flagellar biosynthesis genes flhA, flhB2 and fliR of Leptospira interrogans and construct their prokaryotic expression system. Methods Extract genomie DNA from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain 56601 by phenol-chlorofom method and amplify flhA,flhB2 and fliR gene fragments by high fidelity PCR. After T-A cloning, the amplified genes were identified by sequencing and used for the construction of prokaryotic expression vector. Identify the expressed fusion protein by SDS-PAGE and Western blot. Results The homologies of nucleotide sequences of amplified flhA, flhB2 and fliR genes to those reported were 100% ,99. 9% and 99. 9% ,and those of the deduced amino acid sequences were 100% ,100% and 99. 8% ,respectively. Fusion proteins Trx-FlhA ,Trx-FlhB2 and Trx-FliR were effectively expressed in the constructed prokaryotic expression system under induction of IPTG. The expressed product contained about 10% of total somatic protein. Conclusion The prokaryotic expression system of flagellar biosynthesis genes flhA, flhB2 and fliR of Leptospira interrogans was successfully constructed.