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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 ,抑制了HBe/HBcAg的表达
OBJECTIVE: To observe the effect of double-stranded ribozyme against HBV C gene blockade on C cell gene expression. Methods: The double-stranded ribozyme fragment was excised from pGEM-Rz12 3 (containing double-site ribozyme targeting HBVC region) by subcloning technique and was cloned into eukaryotic expression vector pBBS2 12. The recombinant plasmids pBBS2 12 -Rz and pBBS2 12 were transfected into 2115 cells by lipofectamine, and the expression of ribozyme was observed by dot blot hybridization. ELISA, immunofluorescence, immunohistochemistry, image analysis, Western blot analysis HBe / HBcAg and HBsAg expression. Results: After transfected 2 215 cells were screened by hygromycin B and G418 for 2 weeks, dot blot hybridization confirmed that the two-site ribozyme against HBVC region could be expressed. The results of enzyme-linked immunosorbent assay showed that ribozyme inhibited the expression of HBeAg by 48.6%. Immunofluorescence, immunohistochemistry, image analysis and Western blot analysis confirmed that ribozyme could inhibit the expression of HBV in vivo. Conclusion: The double-site ribozyme can block the expression of C gene and block the expression of HBe / HBcAg by cutting against the gene of HBV C region