A Study on Construction of Plasmid pcDNA3.1/HA-myc-His(-)Z-CD and Transfection into HEK93 Cell

Objective:To construct a recombinant plasmid containing the suicide gene CD and study the expression of CD gene in HEK293 cert.Methods:A fragment containing full-lengh coding region of CD was subcloned into HAtag-tagged vector pcDNA3.1/HA-myc -His(-)Z to construct recombinant plasmid pcDNA3,1/HA-myc-His(-)Z-CD.CD was identified by enzyme digestion of EcoRI/ BamHI and sequence,then rite positive recombinant plasmid was transfected into HEK293 cells using a routine lipofectamine method.After 48h, total protein was extracted and the expression of the CD gene in transfected HEK293 cells was identified by western blot.Results:A fragment of 5.5kb and inserted fragment of 1.3kb were got by cutting positive recombinant plasmid of pcDNA3.1/HA-myc-His(-)Z-CD with EcoRI/ BamHI.Automatic DNA sequence analysis demonstrated that sequence of the recombinant plasmid pcDNA3.1/HA-myc-His(-)Z-CD was totally the same with that published in GenBank.The expression of CD gene was detected by western blot.Conclusion:pcDNA3.1/HA-myc- His(-)Z-CD was successfully constructed.