阴道毛滴虫黏附蛋白33基因真核表达载体的构建与鉴定

Construction of Trichomonas vaginalis Adhesion Protein 33 Gene Eukaryotic Expression Plasmid

目的构建并鉴定阴道毛滴虫黏附蛋白33基因(adhesionprotein33gene,ap33gene)真核表达载体。方法提取阴道毛滴虫分离株基因组DNA,PCR扩增黏附蛋白33基因,克隆入pMD-18T线性质粒,重组子经双酶切、PCR鉴定及测序分析。鉴定出的重组质粒pMD-18T-ap33和pcDNA3.1(+)空质粒经BamHⅠ和XbaⅠ限制性内切酶双酶切,凝胶电泳后回收ap33目的基因和pcDNA3.1(+)空质粒酶切片段,将ap33基因亚克隆入pcDNA3.1(+)载体并进行筛选和鉴定。结果PCR扩增出阴道毛滴虫黏附蛋白33基因,重组质粒pMD-18T-ap33经双酶切,PCR及序列分析,ap33基因的长度为930bp,与GenBank上公布的ap33基因序列同源性达99%。经凝胶电泳、PCR鉴定和限制性酶切鉴定,构建出pcDNA3.1(+)-ap33重组质粒。结论成功构建了阴道毛滴虫pMD-18T-ap33克隆质粒及pcDNA3.1(+)-ap33重组质粒。

Objective To construct and identify the Trichomonas vaginalis adhesion protein 33 （ap33 gene） eukaryotic expression plasmid. Method Total DNA was extracted from Trichomonas vaginalis isolate. The ap33 gene amplified by PCR was cloned into pMD-18T vector. The recombinant plasmid pMD-18T-ap33 was identified by PCR and restriction analysis, and the insert was sequenced. The plasmid pMD-18T-ap33 and pcDNA3.1 （＋） were digested by BamH I and Xba I. The ap33 gene was subcloned into the plasmid pcDNA3.1 （＋）. The recombinant plasmid pcDNA3.1 （＋）-ap33 was identified by PCR and restriction analysis. Result The ap33 gene was amplified by PCR technique. The recombinant plasmid pMD-18T-ap33 was constructed and conftrmed by PCR and restriction analysis. The size of ap33 gene was 930bp, which had 99% of homology with the sequence of ap33 gene published in GenBank. The PCR and restriction analysis proved the recombinant plasrnid pcDNA3.1 （＋）-ap33 was correctly constructed. Conclusion The plasmid pMD-18T-ap33 and the eukaryotic expression plasmid pcDNA3.1 （＋）-ap33 were constructed successfully.