腺病毒介导核酶对细胞内HBV基因表达的抑制作用

目的观察针对HBV(乙型肝炎病毒)C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HBV C区双位点核酶)上切下双位点核酶的片段,定向克隆于腺病毒表达载体pAd-CMV中。利用Lipofectamine2000介导,将重组病毒pAdCMV—Rz及pAdCMV—laz感染2.2.15细胞中,采用打点杂交技术观察核酶的表达,采用ELISA(酶联免疫)、免疫荧光、共聚焦定量、图象分析法、Western blot分析Hbe/HBcAg的表达。结果感染的2.2.15细胞2周后,打点杂交证实可表达针对HBV C区双位点核酶。ELISA方法检测发现核酶抑制HBeAg表达68.6％,用免疫荧光、免疫组化、图象分析法、Western blot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HBV C区基因的剪切作用,阻断C区基因表达,抑制了HBe/HBcAg的表达。

The adenovirus expression ribozyme inhibits HBV expressed in cell

Objective To observe thd inhibition of HBc/HBeAg expressed in the 2.2.15 cell infected by two-unit ribozyme. METHODS By use of subclone technique, the two-unit ribozyme gene which cut from pGEM - Rzl23 was ligated into the adenovirus expression vector pAdCMV. The recombinant adenovirus was infected into the 2.2.15 cell. The ribozyme was detected by dot - blot hybridization . The e/c antigen of HBV were detected by ELBA, immunohistochemical technology, image analysis system and western blot. RESULT We found that the ribozyme expressed in the 2.2.15 cells by dot - blot hybridization. The level of HBeAg can be reduced by up to 68.6% in the infected 2.2.15 cell with two- ribozyme. By using inmunohistochemical technology and image analysis system, western blot, we found the level of HBcAg expressed in endocellular went down. CONCLUSION All the results strongy indicate that the two - unit ribozyme can inhibit the HBV expression in the cell.