Construction of double-copy and x gene deleted hepatitis B virus DNA expression plasmid and cells transfection study

Objective:To construct double copy and x gene deleted HBV expression plasmid and study its expression in Hep3B cell line. Methods:The double copy HBV DNA ( adr Ⅰ) was used to inactivate HBV x gene by inserting mutation and gene recombination. The inserted 55 bp DNA sequence was synthesized artificially; the insertion point was ApaL Ⅰ of x gene area. After recombination, an x gene defected HBV plasmid containing single P, S and C gene was constructed,which can express in mammalian cell line. Another plasmid carrying double copy HBV DNA with normal x gene was constructed as contrast. Both were used to transfect Hep3B cells. Then the cells were screened by G418 and HBV virus in culture medium were isolated and detected by fluorescence quantitative PCR. Results: Plasmids pcDNA3 KN F1F2 and pcDNA3 ES HBV2 were constructed successfully. After cell transfection, the HBV DNA was highly expressed with both plasmids on the 3 rd, 6 th,14th day. Conclusion: The plasmids constructed can express in Hep3B cell line and cause HBV replication; x gene defected HBV gene has no effect on HBV replication in Hep3B cell line.