腺相关病毒载体介导siRNA抑制HBV的表达与复制

中国生物工程杂志

2010, Vol. 30

Issue (04): 49-53

研究报告

腺相关病毒载体介导siRNA抑制HBV的表达与复制

辛娟娟¹,孟庆玲²,高向东¹,刘金毅^3**

1.中国药科大学生命科学与技术学院南京 210009
2. 中国疾病预防控制中心病毒病预防控制所北京 102206
3.北京三元基因工程有限公司北京 102600

Inhibition of Hepatitis B Virus by Small Interfering RNA Expressed from Adenovirus-associated Virus Vectors

XIN Juan-juan¹,MENG Qing-ling²,GAO Xiang-dong¹,LIU Jin-yi³

1.School of life Science & Technology, China Pharmaceutical University, Nanjing 210009,China
2.Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
3.Beijing Tri-Prime Genetic Engineering Co. Ltd, Beijing102600, China

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摘要：

目的：构建并制备能够有效抑制HBV mRNA表达的siRNA重组腺相关病毒。方法：将筛选到的siRNA片段克隆入骨架质粒pAAVMCS中，与pAAVRC和pHelper质粒共转染人胚肾293T细胞，包装出重组腺相关病毒，将纯化后的重组病毒直接感染Hep G 2.2.15细胞，通过酶联免疫法和荧光定量PCR法检测重组腺相关病毒的抑制效果。结果：重组腺相关病毒显著降低HBV 分泌的相关抗原蛋白以及DNA和RNA水平。结论：重组腺相关病毒载体介导的siRNA能够有效抑制HBV的表达与复制。

关键词： RNA干扰; HBV; 腺相关病毒; 基因治疗

Abstract:

Objective: To construct the recombinant Adenovirus-associated virus(AAV) vector for siRNA targeting HBV mRNA and to evaluate the inhibitive effect on Hep G 2.2.15 cell. Method: The packing cell line(human embryonic kidney 293T cell) was co-transfected with AAV backbone plasmid pAAV-MCS which has been cloned into the selected siRNA , along with the pAAV-RC and pHelper in AAV Helper-Free System. The recombinant Adenovirus-associated virus was packaged and amplified, followed by infection of the Hep G 2.2.15 cells. The expression level of HBV gene and the replication of HBV were analyzed by ELISA and fluorescence quantitative RCR. Result: The recombinant Adenovirus-associated virus vectors containing siRNA targeting HBV mRNA was successfully constructed. ELISA results demonstrated that the siRNA can effectively inhibit the secretion of HBsAg and HBeAg and the fluorescence quantitative RCR results also showed that the copies of HBV DNA and RNA were significantly reduced. Conclusion: The recombinant Adenovirus-associated virus vector can effectively inhibit the expression and replication of HBV in vitro.

Key words: RNA interference HBV Adenovirus-associated virus Gene therapy

收稿日期: 2010-02-26 出版日期: 2010-04-29

通讯作者: 刘金毅 E-mail: liujy1207@sina.com.cn

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引用本文:

辛娟娟孟庆玲高向东刘金毅. 腺相关病毒载体介导siRNA抑制HBV的表达与复制[J]. 中国生物工程杂志, 2010, 30(04): 49-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I04/49

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