Construction of Eukaryotic Exression Vector for Hydrogenosomal Adenynate Kinase(AK) of Trichomonas Vaginalis

Objective To construct and identify eukaryotic expression vector of the hydrogenosomal adenynate kinase of Trichomonas vaginalis. Method A pair of primers was designed according to the known sequence of AK gene. The AK gene fragment was amplified by PCR,and ligated to a sequenceing vector,pMD18—T simple vector.Positive recombinants were detected by PCR,digested by restriction enzyme and sequenced with dideoxy nucleotide chain termination method. The cloning vectors( containing full length of AK DNA and two restriction sites:BamHI and XbaI)were first cut by two restriction andonuclease:BamHI and XbaI,and the same as the eukaryotic expression vector;then,the AK DNA and the digested vector were ligated by T4 DNA ligase,and rcombinant rukaryotic expression vector was formed. Its length was certificated by agarose gel electorphoresis analysis,digestion with BamHI and XbaI,and PCR. Result The results of agarose gel electrophoresis analysis,digestion,and PCR confirmed the right length of inserted DNA,which was the same as the AK DNA. Conclusion pcDNA3.1-AK,a eukaryotic expression vector,is successfully constructed.