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

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。

Cloning of Flagellar Biosynthesis Genes flhA, flhB2 and fliR of Leptospira interrogans and Construction of Prokaryotic Expression System

Objective To clone the flagellar biosynthesis genes flhA,flhB_2 and fliR of Leptospira interrogans and construct their prokaryotic expression system.Methods Extract genomic DNA from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain 56601 by phenol-chloroform method and amplify flhA,flhB_2 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,flhB_2 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-FlhB_2 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,flhB_2 and fliR of Leptospira interrogans was successfully constructed.