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目的探讨寄生性原虫阴道毛滴虫细胞生长和衰老的相关基因。方法从阴道毛滴虫的cDNA表达文库中分离出两个与酵母沉寂信息调节因子(Sir2)有较高同源性的cDNA克隆,分别命名为TvSir2和TvSir2-like,它们的编码框分别长915bp和1116bp。结果序列分析显示这两个cDNA克隆与酵母Sir2同源性很高,其氨基酸序列中含有Sir2p及其同源蛋白三个特征性保守结构域。分别从这两株cDNA克隆中扩增出表达片段植入表达载体pET-41a,转化宿主菌E.coli BL21并用IPTG(isopropylthio-β-D-galactoside)诱导表达到大量融合蛋白。用亲和层析法纯化的融合蛋白分别免疫豚鼠,获得的抗血清用Western-blot法识别到滴虫虫体全蛋白中大小为34000Mr和42000 Mr的条带。免疫荧光法检测TvSir2和TvSir2-like蛋白位于细胞核外的内质网和高尔基复合体区域。结论TvSir2和TvSir2-like克隆是酵酶Sir2的同源基因,为TvSir2和TvSir2-like在模式生物阴道毛滴虫的功能研究奠定了基础。
Objective To investigate the genes involved in the growth and senescence of Trichomonas vaginalis cells. Methods Two cDNA clones with high homology with yeast silent regulator (Sir2) were isolated from the Trichomonas vaginalis cDNA library and named as TvSir2 and TvSir2-like, with the coding sequences of 915bp and 1116bp . Results Sequence analysis showed that these two cDNA clones have high homology with yeast Sir2, and their amino acid sequences contain three characteristic conserved domains of Sir2p and its homologous proteins. The expression vector pET-41a was amplified from the two cDNA clones and transformed into host E.coli BL21. The fusion protein was induced with IPTG (isopropylthio-β-D-galactoside). Guinea pigs were respectively immunized with the fusion protein purified by affinity chromatography. The obtained antiserum was identified as bands of 34000Mr and 42000 Mr in the whole protein of Trichomonas vaginalis by Western-blot. TvSir2 and TvSir2-like proteins were detected by immunofluorescence in the regions of endoplasmic reticulum and Golgi complex outside the nucleus. Conclusion TvSir2 and TvSir2-like clones are homologous genes of yeast Sir2, which lays the foundation for the study on the function of TvSir2 and TvSir2-like in the model organism Trichomonas vaginalis.