阴道毛滴虫黏附蛋白65-3基因的cDNA克隆与序列分析

目的对阴道毛滴虫黏附蛋白65-3基因进行cDNA克隆与序列分析。方法Trizol试剂提取阴道毛滴虫基因组RNA。以总RNA为模板,RT-PCR扩增黏附蛋白65-3基因,定向克隆入pMD-18T线形载体,构建pMD-18T-ap65-3重组质粒,并进双行酶切﹑PCR鉴定及测序分析。结果RT-PCR扩增出阴道毛滴虫黏附蛋白65-3基因。经凝胶电泳﹑PCR鉴定和限制酶切鉴定,成功的构建出pMD-18T-ap65-3重组质粒。序列分析表明,黏附蛋白65-3基因开放阅读框长为1 704 bp,与GenBank上公布的黏附蛋白65-3基因序列比较,其同源性高达99.6%。结论阴道毛滴虫黏附蛋白65-3基因体外扩增及测序的成功,为进一步研究阴道毛滴虫的致病机理及滴虫病防治方法奠定了基础。

Cloning and Sequence Analysis of Trichomonas vaginalis Adhension Protein 65-3

Objective To clone and sequence the cDNA coding for the adhensin protein 65-3 of Trichomonas vaginalis(ap65-3 gene).Method Total RNA was extacted from Trichomonas vaginalis 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 inserted fragment was sequenced.Result The ap65-3 gene was amplified from total RNA by RT-PCR.The recombinat plasmid pMD-18T-ap65-3 was constructed and comfirmed by PCR and restriction analysis.The sequence analysis revealed that ap65-3 gene contains an open reading frame with 1 740 bp and has 99.6% homology with the sequence of ap65-3 gene published on GenBank.Conclusion The ap65-3 gene was cloned and sequcenced succesfullly.The gene can be used in the future analysis of the pathogenic mechanisms of Trichomonas vaginalis and for the development of treatment for trichomoniasis.