双位点核酶抑制细胞内乙型肝炎病毒表达的研究

Intracellular application of two unit ribozyme gene against hepatitis B virus

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 核酶 (Rz) 12 3 (含针对HBVC区 2 0 2 9～ 2 0 3 1及 2 0 63～ 2 0 65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pBBS2 12中 ,利用脂质体介导 ,将重组质粒pBBS2 12 Rz与p1.2Ⅱ (含乙型肝炎病毒全序列 )共转染入人肝癌细胞 (HHCC)中 ,采用原位杂交观察核酶的表达 ,ELISA、免疫荧光、激光共聚焦、免疫组化及图像分析法分析HBeAg/HBcAg及HBsAg的表达。结果 共转染HHCC后第 5天上清中测到HBsAg ,第 7天测到HBeAg ,筛选后达高峰 ,上清中HBsAg 1∶10 0阳性 ,胞浆裂解液中HBcAg 1∶4阳性 ,且可稳定表达 2个月以上。共转染HHCC经潮霉素B筛选 2周后 ,原位杂交证明可表达针对HBVC区双位点核酶。ELISA方法检测发现 ,核酶抑制HBeAg表达 5 0 %～ 65 % ,用免疫荧光、激光共聚焦分析 ,免疫组化等均证实核酶可抑制HBV细胞内的表达。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HBeAg/HBcAg的合成。

Objective To observe the inhibition of HBeAg/HBcAg expressed in the human hepatic carcinome cellular (HHCC) line transfected by two unit ribozyme. Methods By using subclone technique, the two unit ribozyme gene which was cut from pGEM Rz123 was ligated into the eukaryotic expression vector pBBS212. The recombinant plasmid pBBS212 Rz and p1.2Ⅱ plasmid carrying genome of adr subtype HBV, were cotransfected into the HHCC using lipofectamine. The ribozyme was detected by in situ hybridization. The HBsAg and HBeAg/HBcAg were detected by ELISA, immunohistochemical technology and image analysis system and laser confocal imaging system anlysis. Results The cotransfection cell can express HBsAg and HBeAg/HBcAg. After the transfected cell was selected for two weeks by hygromycin B, the ribozyme can express on the HHCC by in situ hybridization. The level of HBeAg can be reduced by up to 50%～65% in the cotransfected HHCC with two unit ribozyme. By using immunohistochemical technology and image analysis system and laser confocal imaging system anlysis, the level of HBcAg endocellular expressed dropped down. Conclusion All these results strongly indicate that two unit ribozyme can inhibit the HBV expression in the cell.