逆转录病毒载体介导双靶区乙型肝炎病毒反义RNA模型的构建

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 recombinant retrovirus vector carrying dual target antisense RNA of hepatitis B virus

To probe the ways by which the antisense gene of HBV can be transcribed in eukaryotic cell to inhibit HBV replication and expression,the recombinant retrovirus vector carrying dual target antisense RNA of hepatitis B virus was constructed. Antisenses of HBV subtype ayw X region and P region were constructed by artificial synthesis and were named the X and P fragments.The X and P fragments were inserted into the retrovirus pLXSN vector cloning site in the sense or antisense orientation respectively with recombinogenic technique.Then recombinant retrovirus vector carrying sing target antisense RNA was constructed successfully.Construction of recombinant retrovirus vector carrying dual target antisense RNA of HBV is in the foundation of construction of recombinant retrovirus vector carrying sing target in similar ways.The recombinant retroviurs vector carrying dual target antisense RNA of hepatitis B virus had been constructed successfully and it had been authenticated by restriction enzyme digestion and electrophoretic analysis fragments of.PCR amplification and DNA sequence code were checked with automatic sequenator.The recombinant retrovirus vector carrying dual target antisense RNA of hepatitis B virus will be a good foundation for studying the inhibition of variable HBV in eukaryotic cell and for the antivirus treatment of polygenic disease.