双位点核酶对2.2.15细胞中HBV基因表达抑制作用

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

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 -Rz12 3(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pBBS2 12中。利用lipofectamine介导 ,将重组质粒pBBS2 12 -Rz及pBBS2 12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 。

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.