阴道毛滴虫Rab11鸟苷三磷酸酶cDNA克隆和序列分析(英文)

Molecular Cloning and Sequence Analysis of Rab11 GTPase in Trichomonas vaginalis

目的近年研究表明Rab11鸟苷三磷酸酶在调节各种真核细胞的膜转运通道中发挥着重要作用。但是,对于原生动物毛滴虫膜转运系统的研究仍甚少。本研究旨在克隆和分析阴道毛滴虫Rab11鸟苷三磷酸酶基因,以便进一步探讨其功能。方法使用SMARTcDNA文库构建试剂盒构建阴道毛滴虫cDNA表达文库,用生物信息学进行TvRab11同源分析,鉴定TvRab11基因,用PCR方法扩增基因组DNA,TA克隆TvRab11cDNA、测序及序列分析。结果在分离出的cDNA克隆中发现一个Rab11鸟苷三磷酸酶同源基因。该cDNA序列长710bp,读码框含636bp,推测蛋白质序列具211个氨基酸。序列分析表明该基因系Rab11亚家族的同源基因,其推测肽链与拟南芥Rab11c的同源性最高(一致性60%,相似性79%),与人类Rab11b次之(一致性58%,相似性78%)。该氨基酸序列拥有Rab鸟苷三磷酸酶家族的所有保守结构域和特异性Rab结构域(RabF1-5)。进化树分析也表明该基因系Rab11的同源基因。序列分析还显示该基因的基因组DNA序列与cDNA序列完全一致,提示该基因无内含子。结论分析表明该基因是阴道毛滴虫Rab11鸟苷三磷酸酶同源基因,其功能有待进一步研究。

Objective Rab11 GTPases play an essential role in regulating membrane trafficking pathways in eukaryotic ceils. Nonetheless, there has been little work done on characterizing the transport machinery of Trichomonas. The aim of this study is to clone and characterize a Rabll gene of Trichomonas vaginalis. Methods A cDNA expression library was constructed with T.vaginalis total RNA. A cDNA clone, which showed a high degree of homology with Rab proteins of different species, was isolated and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and ClustalW programs. The genomic DNA corresponding to the eDNA sequence was amplified using PCR techniques and following by sequencing. Results eDNA with a length of 710 base pairs and an open reading frame of 636 bp was obtained. The deduced amino acid sequence from the open reading frame was found to possess 211 residuals. Sequence analysis demonstrated that this cDNA clone was homologous to the Rab11 subfamily of different species （60% identity and 79% similarity with Arabidopsis thaliana Rabllc, 58% identity and 78% similarity with human Rabllb）, and that the amino acid sequence contains all the well known conserved sequence elements of Rab family. Specific Rab motifs were also detected in the deduced amino acid sequence. Phylogenetie analysis showed that its closest homologues are Rabll proteins from other species. Sequencing of the PCR product of genomie DNA revealed that the genomie DNA sequence encompassing the putative 5＇-ATG and 3＇-stop eodon is identical to the eDNA sequence. Conclusion A eDNA clone corresponding to the T-vaginalis Rabll gene was obtained.The function of this gene in regulating membrane trafficking pathways of the parasitic protist is still under investigation.