反义基因转移表达及抗乙型肝炎病毒的作用

目的观察重组逆转录病毒载体－包装细胞系统介导反义基因转移表达和抗乙型肝炎病毒（ＨＢＶ）的作用。方法将ＨＢＶａｙｗ１４０２－２９０６片段和２８３９－１９８６片段反向插入重组逆转录病毒载体质粒，分别转染ＰＡ３１７包装细胞，分别感染ＮＩＨ３Ｔ３细胞和２．２．１５细胞。结果转导的ＮＩＨ３Ｔ３细胞内有ＨＢＶ反义ＲＮＡ转录表达。ＨＢＶ反义基因重组逆转录病毒感染２．２．１５细胞后第３天，表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ）表达量减少，感染后第５天，ＨＢＶ抗原表达抑制率达高峰。ｐｒｅＳ／Ｓ片段反义基因转移后，ＨＢｓＡｇ表达抑制率为７１％，ＨＢｅＡｇ抑制率为２３％；ｐｒｅＣ／Ｃ片段反义基因转移后，ＨＢ－ｓＡｇ表达抑制率为２３％，ＨＢｅＡｇ抑制率为５９％。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内转移和表达，并对ＨＢＶ复制和表达均有抑制作用。

Transfer and expression of antisense genes of hepatitis B virus (HBV) and their anti HBV effects

Objective To observe the transfer and expression of HBV antisense genes and their anti HBV effects in 2.2.15 cells mediated by retroviral vector packaging cell line system.Methods The recombinant retroviral vectors mediating expression of HBV antisense RNA complementary to 1402-2906 fragment (preC/C) or 2839 1986 fragment (preS/S) of HBV DNA were used to transduce into NIH 3T3 cells and 2.2.15, respectively. Then, the total RNAs of tranduced NIH 3T3 cells were extracted and hybridized with HBV DNA probe. The HBsAg and HBeAg in the supernatants of cultured 2.2.15 cells were assayed with RIA method and the DNAs were hybridized with HBV DNA probe by dot blot.Results The results showed that the HBV antisense genes could be transfered into and expressed in the NIH 3T3 cells mediated by recombinant retroviral vector packaging cell line (PA317 cell). The inhibitory effects on the expression of HBV antigens by antisense RNAs appeared as early as on the day 3 after transduced, reached peak level on the day 5, and persisted at least for eleven days. The inhibtory rates of HBsAg and HBeAg were 71% and 23% by antisense preS/S, and 23% and 59% by antisense preC/C on the day 5 after transduction. In comparison to blank control or sense-gene expressing vectors, the inhibitory effects of HBV antisense vectors were highly significant ( P <0.01). HBV DNA level in the supernatant of the 2.2.15 cells transduced with either antisense preS/S or preC/C in comparison to the blank control vectors was also reduced on the day 5 after transduction as detected by DNA dot blot assays, but the viability of transduced 2.2.15 cells was not affected as detected by MTT assays.Conclusions HBV antisense genes can be transfered and expressed in the eukaryotic cells and the expression of HBV antisense RNAs in 2.2.15 cells can inhibit the replication and expression of HBV.