阴道毛滴虫粘附蛋白65-3基因真核表达载体的构建与鉴定

目的构建并鉴定阴道毛滴虫粘附蛋白65-3基因的真核表达载体。方法Trizol试剂提取阴道毛滴虫株基因组总RNA，RT-PCR扩增粘附蛋白65-3基因，定向克隆入pMD-18T线性质粒，重组子经双酶切、PCR鉴定及测序分析。鉴定正确的重组质粒pMD-18T-ap65-3和peDNA3．1（．＋）空质粒径BamHI和XhoI限制性内切酶双酶切后，凝胶电泳纯化回收目的片段，将粘附蛋白65-3基因亚克隆入peDNA3．1（＋）载体并进行筛选和鉴定。结果成功克隆出阴道毛滴虫粘附蛋白65-3基因，序列分析发现该基因的开放阅读框长为1704bp，与GenBank上公布的粘附蛋白65-3基因序列比较，同源性高达99．6％，经PCR鉴定，限制性酶切鉴定及测序鉴定，正确构建出重组质粒peDNA3．1-ap65-3。结论成功克隆出粘附蛋白65-3基因并构建出真核表达质粒pcDNA3．1-ap65-3，为进一步研究该基因的功能及滴虫的致病机制奠定实验基础。

Construction and identification of trichomonas vaginalis adhesion protein 65-3 gene eukaryotic expressin plasmid

Objective To eonstruee and identify the Trichomonas vaginalis adhesion protein 65-3（ab65-3 germ）eukaryotie expression plasmid. Methods Total RNA was extracted from Trichomonas vagonalis isolate using Trizol reagent. The ap65-3 gene amplified by RT-PCR was cloned into pMD-18T vector. The recombinant plasmid pMD-18T-ap65-3 was identified by PCR and restriction analysis,and the insert fragment was ,sequeneed.The plasmid pMD-18T-ap65-3 and peDNA3.1（ ＋ ）were digested by BamH I and Xho I,Then the ap65-3 gene was subcloned into the plasmid peDNA3.1 （ ＋ ） .The recombinant plasmid peDNA3.1-ap65-3 was identified by PCR-restriction analysis and sequencing.Results The ap65-3 gene was cloned suecessfully,The sequence analysis revealed that ap65-3 gene contains an open reading frame with 1804 bp and had 99.6% of homology with the sequence of ap65-3 gene published in Gen Band.The PCR, restriction analysis and sequencing proved the recombinant plasmid peDNA3.1 -ab65-3 was correctly constructed. Conclusion The ap65-3 gene was cloned and eukaryotie expresssion plasmid peDNA3.1-ap65-3 wae constructed suceessfttUy. The works were helpful for studies on the pathogenisis of Trichomonas vaginalis and function of the ap65-3 germ.