双位点核酶对2.2.15细胞中HBV基因表达抑制作用

目的:观察针对HBVC区双位点核酶在细胞内阻断C区基因表达的作用。方法:采用亚克隆技术,从pGEM-Rz123(含针对HBVC区双位点核酶)上切下双位点核酶的片段,定向克隆于真核表达载体pBBS212中。利用lipofectamine介导,将重组质粒pBBS212-Rz及pBBS212转染2.2.15细胞中,采用打点杂交技术观察核酶的表达,采用ELISA、免疫荧光、免疫组化、图象分析法、Westernblot分析HBe/HBcAg及HBsAg的表达。结果:转染的2.2.15细胞经潮霉素B和G418筛选2周后,打点杂交证实可表达针对HBVC区双位点核酶。ELISA方法检测发现核酶抑制HBeAg表达48.6%,用免疫荧光、免疫组化、图象分析法、Westernblot分析证实核酶可抑制HBV细胞内表达。结论:该双位点核酶通过针对HBVC区基因的剪切作用,阻断C区基因表达,抑制了HBe/HBcAg的表达。

Two-unit ribozyme inhibit hepatitis B virus expressed in 2.2.15 cell

AIM: To observe the inhibition of HBc/HBeAg expression in the 2.2.15 cell transfected by two-unit ribozyme. METHODS: By use of subclone technique, two-unit ribozyme gene which was cutted from pGEM-Rz123 was ligated into the eukaryotic expression vector pBBS212. The recombinant plasmid was cotransfected into 2.2.15 cell using lipofectamine. Ribozyme was detected by dot-blot hybridization. The s and e/c antigen of HBV were detected by using ELISA, immunohistochemical technique, image analysis system and Western blot. RESULTS: After the transfected cell was selected two weeks by hygromycin B and G418. We found by dot-blot hybridization that ribozyme can express on 2.2.15 cell. HBeAg level can be reduced by 48 6% in the transfected 2 2 15 cell with two-ribozyme. Using immunohistochemical technique and image analysis system, Western blot, we observed that the level of HBcAg expressed in endocellular went down. CONCLUSION: All these results strongly indicate that two-unit ribozyme can inhibit hepatitis B virus expression in the cell.