阴道毛滴虫Rab1a基因的cDNA克隆及重组表达

目的克隆和分析阴道毛滴虫(Trichomonasvaginalis,Tv)Rab1a基因,构建Rab1a基因表达重组载体并表达其融合蛋白,以进一步探讨其功能。方法提取阴道毛滴虫基因组DNA为模板,扩增TvRab1a基因,用pQE80L载体与TvRab1acDNA克隆构建原核表达重组体并表达融合蛋白,纯化表达产物并由SDS-PAGE鉴定。结果在阴道毛滴虫cDNA文库克隆中发现了Rab1a基因,序列分析显示Rab1a基因的基因组DNA序列含有一个25bp大小的内含子。成功构建了pQE/Rab1a原核表达重组体,并表达出预期大小的重组蛋白质。结论分析表明TvRab1a基因是阴道毛滴虫Rab1鸟苷三磷酸酶同源基因,它含有一个25bp的内含子。获得了该基因的重组蛋白,将对TvRab1a基因的功能进行进一步研究。

Cloning and Expression of A Rab1a Gene from Trichomonas vaginalis

Objective To clone and characterize the Rab1a gene of Trichomonas vaginalis. Method The cDNA clone of Rab1a was isolated from Tv cDNA expression library and sequenced. The predicted amino acid sequence shows high homology with Rab proteins of different species. Sequence analysis was performed. The genomic DNA was amplified using PCR techniques and sequenced. The recombinant plasmid of pQE/TvRab1a was constructed, and the fusion protein was expressed in E.coli M15. Result The Rab1a gene was isolated and sequenced. Sequence analysis demonstrated that this cDNA clone was Rab1a isoform. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA contains an intron of 25 bp.The recombinant plasmid pQE/TvRab1a was constructed, and a fusion protein was expressed in E.coli M15. Conclusion These data suggest that the cDNA clone is encoding the Rab1a of T.vaginalis. The T.vaginalis Rab1a gene has an 25 bp intron.