逆转录病毒载体介导乙型肝炎病毒反义基因的转录表达

Transcription of antisense RNA of hepatitis B virus through retroviral mediated gene transfer

为了探索在真核细胞内转录表达乙型肝炎病毒（ＨＢＶ）反义核酸的方法，用基因重组技术将ＨＢＶ前Ｃ／Ｃ基因（ＰｒｅＣ／Ｃ）和前Ｓ／Ｓ基因（ＰｒｅＳ／Ｓ）片段反向插入逆转录病毒载体质粒，再将重组体分别转染ＰＡ３１７包装细胞，进而获得能够介导ＨＢＶ反义基因向小鼠ＮＩＨ３Ｔ３细胞转移表达的重组逆转录病毒。经分子杂交试验表明，含有ＨＢＶ反义基因的重组逆转录病毒序列已经整合到转染的ＰＡ３１７细胞染色体上；转导的ＮＩＨ３Ｔ３细胞内有ＨＢＶ反义ＲＮＡ转录表达。结论：逆转录病毒载体包装细胞系统能够介导ＨＢＶ反义基因在真核细胞中转录表达。

To probe the ways by which the antisense gene of hepatitis B virus (HBV) can be transferred and transcripted in eukaryotic cells to inhibit HBV replication and expression. Two retroviral vectors that carried antisense gene of hepatitis B virus (HBV) PreC/C or PreS/S region were constructed. The HBV ayw PreC/C and PreS/S fragments were inserted into the vector pDO.R cloning site in the sense or antisense orientation and the recombinant retroviral vectors were then transfected into PA317 packaging cells by calcium phosphate coprecipitation, respectively. The stably transformed G418resistant PA317 cells were selected and the recombinant retroviruses released from transfected G418resistant PA317 cells were assayed by G418 selection method, using NIH 3T3 cells as target cells. Southern blot and RNA Dot blot analysis showed that the recombinant retroviral vector sequences were stably integrated into the chromosome of transfected PA317 cells and the antisense RNA of HBV PreS/S or PreC/C gene also transcripted in the transduced NIH 3T3 cells. These results suggested that antisense gene of HBV can be successfully transferred and transcripted in target cells through retroviral vector mediated gene transfer and the antisense retroviral vectors may be potentially useful for antiHBV gene therapy.