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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。
In recent years, studies have shown that Rab11 guanosine triphosphatase plays an important role in regulating the membrane transport channels of various eukaryotic cells. However, little research has been done on the protozoan membrane transport system. This study aimed to clone and analyze Trichomonas vaginalis Rab11 guanosine triphosphatase gene in order to further explore its function. Methods The cDNA library of Trichomonas vaginalis was constructed by SMART cDNA library construction kit. The homology of TvRab11 was identified by bioinformatics. The TvRab11 gene was identified by PCR. The genomic DNA was amplified by PCR and sequenced. As a result, a Rab11 guanosine triphosphatase homologous gene was found in the isolated cDNA clone. The cDNA sequence is 710 bp in length and contains 636 bp in reading frame, suggesting that the protein sequence has 211 amino acids. Sequence analysis showed that this gene is a homologue of the Rab11 subfamily which deduced that the peptide chain had the highest homology (Arabidopsis thaliana Rab11c with 60% identity and 79% similarity) to human Rab11b (58% , Similarity 78%). This amino acid sequence owns all the conserved domains and the specific Rab domain (RabF1-5) of the Rab guanosine triphosphatase family. Phylogenetic tree analysis also showed that this gene is a homolog of Rab11. Sequence analysis also showed that the genomic DNA sequence of the gene is exactly the same as the cDNA sequence, suggesting that the gene has no intron. Conclusion analysis showed that the gene is Trichomonas vaginalis Rab11 guanosine triphosphatase homologous genes, its function needs further study.