表达HBsAg特异性siRNA的重组腺相关病毒载体的构建 |
| |
引用本文: | 胡斌,杨燕,刘嘉,马智勇,黄红平,余源,刘慎沛,杨东亮.表达HBsAg特异性siRNA的重组腺相关病毒载体的构建[J].实用肝脏病杂志,2010,13(3):161-165. |
| |
作者姓名: | 胡斌 杨燕 刘嘉 马智勇 黄红平 余源 刘慎沛 杨东亮 |
| |
作者单位: | 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. 实验医学研究中心 |
| |
基金项目: | 国家传染病防治科技重大专项,国家高技术研究发展计划(863)项目,国家自然科学基金 |
| |
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。
|
关 键 词: | HepG2.215细胞 腺相关病毒2 RNA干扰 HBsAg |
Inhibition of HbsAg and HbeAg expression of recombinant adeno-associated virus encoding HBsAg-shRNA in HepG2.215 cells in vitro |
| |
Institution: | HU Bin,YANG Yan,LIU Jia,et al. Institute of Clinical Immunology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China |
| |
Abstract: | 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. |
| |
Keywords: | HBsAg |
本文献已被 CNKI 维普 万方数据 等数据库收录! |
|