阴道毛滴虫两个Sir2同源基因的克隆和功能(英文)

Molecular Cloning and Characterization of Two Hommologues of Yeast Sir2 Gene in the Parasitic Protist Trichomonas vaginalis

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 screen cell growth and senescence-related genes of the parasitic protist Trichomonas vaginalis, we launched an EST program and isolated two cDNA clones from a T.vaginalis cDNA library, which showed high homology in deduced amino acid sequences to yeast Sir2 and designated as TvSir2 and TvSir2-like. Method The cDNA sequence of TvSIR2 had a length of 1034 base pairs （bp） with an open reading frame of 915 bp, and TvSIR2-like, 1214 bp with an open reading frame of 1116 bp. Result The two deduced amino acid sequences shared all the three conserved core domains with yeast Sir2 and its homolognes, suggesting that the two clones were Sir2 homolognes. A cDNA fragment from each cDNA clone was subcloned into the expression vector pET-41a. The expression of the fusion proteins in E.coli BL21 stains was induced by isopropylthio-β-D-galactoside （IPTG）. Two anti-sera were prepared by immunizing two guinea pigs with the purified fusion proteins, Western-blot analysis demonstrated that each anti-serum reacted with the corresponding recombinant protein and detected a clear band （TvSir2,34 000 Mr; TvSir2-like,42 000 Mr） in protein extracts of the protist. Immunofluorescence techniques showed that TvSir2 and TvSir2-like proteins were both localized in the regions of perinuclear （ER） and Golgi complex. Conclusion Our data suggest that TvSir2 and TvSir2-like were two members of Sir2 family. Their biological functions in the protist would be further studied.