Cloning of human vacuolar protein sorting 4A gene and construction of eukaryotic expression vector / 国际检验医学杂志

ABSTRACT

Objective To clone human vacuolar protein sorting 4A gene(hVPS4A)and to construct its eukaryotic expressive plasmid.Methods Primers were designed to amplify the full length hVPS4A by PCR using cDNA of Huh7 cell as a template,then the target DNA was inserted into the eukaryotic vector pRK5.The recombinant plasmid was confirmed by PCR,restriction enzyme digestion and DNA sequencing.Results A 1 300 bp fragment was successfully amplified by PCR from the cDNA of Huh7 cells.Af-ter recycled,purified and ligated with the vector pRK5,the recombinant plasmid was transformed into E.coli DH5α.The positive re-combinant plasmid identified by PCR was selectred and digested by EcoRⅠto get a 5 900 bp fragment;and two fragments including 4 600 bp and 1 350 bp were obtained using EcoRⅠand HindⅢ digestion;the size of these two fragments were consistent with the pRK5 target fragment and the inserted hVPS4A as expected.Moreover,DNA sequencing results confirmed that the inserted frag-ment was in accordance with the hVPS4A reference sequence.Conclusion The eukaryotic expression vector containing hVPS4A gene is constructed successfully,which provides the condition for further study on the hVPS4A biological functions.