表达HBsAg特异性siRNA的重组腺相关病毒载体的构建  

Inhibition of HbsAg and HbeAg expression of recombinant adeno-associated virus encoding HBsAg-shRNA in HepG2.215 cells in vitro

作　　者：胡斌[1] 杨燕[2] 刘嘉[1] 马智勇[1] 黄红平[2] 余源[2] 刘慎沛[2] 杨东亮[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室,武汉市430030 [2]华中科技大学同济医学院实验医学研究中心,武汉市430030

出　　处：《实用肝脏病杂志》2010年第3期161-165,共5页Journal of Practical Hepatology

基　　金：国家传染病防治科技重大专项(2008ZX10002-011);国家高技术研究发展计划(863)项目(2006AA02Z128);国家自然科学基金(30700701)

摘　　要：目的构建表达针对HBsAg小发卡RNA(shRNA)的重组腺相关病毒载体,以观察该病毒在体外对HBsAg和HBeAg的抑制作用。方法将表达针对HBsAg的shRNA定向克隆到pAAV/U6-hrGFP质粒中,获得重组表达质粒(pAAV-shHBs-hrGFP);采用磷酸钙转染法将该质粒与包装质粒pAAV-RC和辅助质粒pHelper共同转染AAV293细胞,进行rAAV-shHBs-hrGFP重组病毒包装。收获病毒感染HepG2.215细胞;采用ELISA法检测HBsAg和HBeAg水平。结果酶切鉴定、测序结果表明,pAAV-shHBs-hrGFP载体成功构建。包装收获的rAAV-shHBs-hrGFP病毒液可以感染HepG2.215细胞,并且可以抑制HBsAg和HBeAg的表达。结论制备的rAAV-shHBs-hrGFP病毒载体能够抑制HBV在体外的抗原表达。Objective To construct the recombinant adeno-associated virus（rAAV）encoding HBsAg-shRNA and to observe its effect on HBsAg and HBeAg expression in HepG2215 cells in vitro. Methods pAAV-shHBs-hrGFP expressing plasmid was constructed by molecular biological techniques. The recombinants were cotransfected with p-RC and p-Helper into AAV-293 cells mediated by calcium acid phosphate. The rAAVs encoding HBsAg-shRNA （rAAV-shHBs-hrGFP）were harvested and infected HepG2215 cells. The silencing effect of this virus on HbsAg and HBeAg gene expression was assessed by ELISA. Results The plasmid of pAAV-shHBs-hrGFP was successfully constructed by identification of restrict enzyme and DNA sequencing. GFP expression was observed about 80% in AAV-293 cells. After cotransfection,the recombinant AAV-shHBs-hrGFP was harvested and the virus titer was tested. The HBsAg and HBeAg expression in HepG2.215 cells was down-regulated markedly after infection of rAAV -shHBs -hrGFP. Conclusion The recombinant AAV -shHBs -hrGFP vector is successfully constructed and can be used as a potential antivirus agent to inhibit HBV replication.

关 键 词：HepG2.215细胞 腺相关病毒2 RNA干扰 HBSAG 

分 类 号：R512.62[医药卫生—内科学]