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

Objective To construce and identify the Trichomonas vaginalis adhesion protein 65-3(ab65-3 gene)eukaryotic expression plasmid.Methods Total RNA was extracted from Trichomonas vaginalis 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 sequenced.The plasmid pMD-18T-ap65-3 and pcDNA3.1(+)were digested by BamH I and Xho I,Then the ap65-3 gene was subcloned into the plasmid pcDNA3.1(+).The recombinant plasmid pcDNA3.1-ap65-3 was identified by PCR-restriction analysis and sequencing.Results The ap65-3 gene was cloned successfully,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 pcDNA3.1-ab65-3 was correctly constructed.Conclusion The ap65-3 gene was cloned and eukaryotic expresssion plasmid pcDNA3.1-ap65-3 wae constructed successfully.The works were helpful for studies on the pathogenisis of Trichomonas vaginalis and function of the ap65-3 gene.