Construction and Identification of HBV X Gene Eukaryotic Expression Vector

Objective: To construct a highly effective eukaryotic expression vector pCDNA3.1 with HBV X gene(pCDNA3.1-X).Methods: HBV DNA was extracted from the HBV DNA positive serum.HBV X gene was amplified by PCR,and identified by gelose electrophoresis analysis.The PCR products and the eukaryotic expression vectors pCDNA3.1 were digested by restriction endonucleases,then the digested X gene was inserted into the digested eukaryotic expression vector. After screening,the positive recombinants were picked out.PCR analysis,Kpn Ⅰ and EcoR Ⅰ restriction endonucleases digest analysis and sequence analysis were used to identify the recombinants.Results: The 480 bp DNA fragment was firstly amplified by PCR from the HBV DNA positive serum.The identical DNA fragment was also amplified from the recombinants.The products of double restriction endonucleases digest analysis were X gene and a 5.5 kb DNA fragment.Sequence analysis showed that the HBV X gene was successfully inserted into pCDNA3.1 vector.Conclusion: The eukaryotic expression vector pCDNA3.1-X has been successfully constructed.