阴道毛滴虫沉寂信息调节因子Sir2同源基因克隆和蛋白表达  

Molecular Cloning and Characterization of A Sir2 Gene in Trichomonas vaginalis

作　　者：史咏梅[1] 傅玉才[1] 徐晓园[1] 许铭炎[1] 刘红[1] 

机构地区：[1]汕头大学医学院寄生虫学教研室,汕头515041

出　　处：《热带医学杂志》2006年第6期611-615,共5页Journal of Tropical Medicine

基　　金：汕头大学研究与发展基金项目(No.L00013)。

摘　　要：目的克隆并分析阴道毛滴虫(Trichomonasvaginalis)沉寂信息调节因子Sir2基因,构建原核表达重组载体,表达重组蛋白,并制备抗体进行相关功能的研究。方法从阴道毛滴虫cDNA表达文库中分离获得阴道毛滴虫Sir2同源基因的cDNA克隆、测序,并采用相关在线程序及软件进行序列分析。将cDNA部分片段亚克隆到原核表达载体pET-41a,转化宿主菌E.coliBL21,IPTG(isopropylthio-β-D-galactoside)诱导表达,亲和层析法纯化表达产物并对表达产物进行SDS-PAGE鉴定之后,用纯化重组蛋白免疫豚鼠获得抗血清,对重组蛋白和滴虫总蛋白进行蛋白质印迹鉴定。结果在阴道毛滴虫cDNA文库中获得一株1034bp的cDNA克隆,序列分析表明,该序列开放阅读框有912bp,推测肽链具有304个氨基酸,等电点(PI)5.5。该肽链与酵母SIR2p蛋白及其同源物一致性高达30%～47%,并具有SIR2p及其同源蛋白的三个高度保守的特征性结构域。进化树结果显示,TvSIR2p接近线虫的SIR2蛋白,与同时克隆到的基因组TvSir2比较序列一致,证明该TvSir2基因组DNA不含内含子。PCR扩增出890bp片段,植入表达载体pET-41a;经酶切鉴定后取阳性克隆用IPTG诱导表达,产生融合蛋白产物经SDS-PAGE鉴定相对分子质量为62000,与预期分子质量相符。用该融合蛋白免疫豚鼠得到的抗体,经Westernblotting检测其与相应融合蛋白发生特异性反应,同时在33000处也能识别滴虫虫体全蛋白。结论推测该克隆是阴道毛滴虫Sir2的同源基因,该基因没有内含子,它可能参与阴道毛滴虫的组蛋白去乙酰化作用及调节衰老和寿命。阴道毛滴虫Sir2基因可以在大肠杆菌中得到表达,并获得了相应的抗血清,为进一步研究其功能奠定了基础。Objective To clone the Trichomonas vaginalis Sir2 gene, express Sir2 recombinant protein and prepare the anti-Sir2 antibodies. Method A cDNA library was constructed with T.vaginalis total RNA. The cDNA clone of TvSir2 was isolated and sequenced.The cDNA sequence of TvSir2 was analyzed using BLASTP, Clastal W and MAGE3 programs, etc. The predicited amino acid sequence shows high homology with Sir2 proteins of differente species. The genomic DNA was amplified using PCR techniques and sequenced. A fragment of the cDNA with the length of 890bp was amplified by PCR and inserted into the expression vector pET-41a, A fusion protein was expressied in E.coli BL21 and purified with Ni-NTA agurosc affinity column. A guinea pig was immunised with the recombinant fusion protein. The antisera collected from the guinea pig were used to identify the fusion protein and the native Sir2 protein by Westren blotting. Result A cDNA clone with a length of 1034 base pairs was isolated and sequenced. The sequence analysis showed that the cDNA clone has an open reading frame with 912 bp. The deduced amino acid sequence contains 304 residues with an estimated pI of 5.5. The predicted amino acid sequence shows homology to Sir2 proteins of different species with 30%～47% identifty. The conserved sequence elements of Sir2, such as the GAG motif, the NID motif and the zinc ribbon were detected in the amino acid sequence. The phylogenetic analysis showed that Trichomonas vaginalis is closer to Caenorhabditis elegans. The genomic DNA of TvSir2 was also amplifyed by PCR and sequenced. Sequence comparison between cDNA and genomic DNA sequence of TvSir2 demonstrated that it is an intronless gene. The recombinant protein was expressed in E.coli, and was used to immunize guinea pig. The antiserum collected from the guinea pig recognized the recombinant protein and detected a clear band of 33 kDa in T.vaginalis protein extracts as expected. Conclusion The cDNA clone isolated is encoding the Sir2 protein. The Trichomonos vaginalis Sir2 gene is intronl

关 键 词：阴道毛滴虫 Sir2克隆 重组蛋白 抗血清 

分 类 号：R382.21[医药卫生—医学寄生虫学]