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构建在真核细胞内转录表达乙型肝炎病毒(HBV)双靶区反义核酸重组载体用以抗HBV基因治疗的研究。方法为合成互补于HBV(ayw亚型)X区核苷酸X片段以及互补于HBV P区的P片段,利用基因重组技术将X、P片段分别正向、反向插入逆转录病毒载体pLXSN的相应酶切位点构建成单靶区重组载体质粒。在构建单靶区载体质粒的基础上,类似方法再构建双靶区重组载体质粒。经酶切电泳、PCR扩增和DNA测序鉴定成功构建了HBV双靶区反义核酸重组载体。为探索在真核细胞内转录表达HBV反义RNA的方法及研究多基因区抗病毒治疗和抗变异病毒打下基础。
Construction of a Recombinant Antisense Vector Transcribed in Hepatitis B Virus (HBV) Double Nuclei Region in Eukaryotic Cells for Anti-HBV Gene Therapy. Methods The X fragment complementary to the X region of HBV (ayw subtype) and the P region complementary to the HBV P region were synthesized, and the X and P fragments were inserted into the retroviral vector pLXSN The corresponding restriction sites were constructed into single-target recombinant plasmid. Based on the construction of single-target vector plasmid, a similar method was used to reconstruct the double-target recombinant vector plasmid. The double-stranded antisense RNA recombinant vector was successfully constructed by restriction enzyme digestion, PCR amplification and DNA sequencing. To lay the foundation for exploring the method of transcribing and expressing HBV antisense RNA in eukaryotic cells and studying multi-gene antiviral therapy and anti-virus.