特异性表达siRNA的新型乙肝核酸药物的研究  

Research of New Hepatitis B's Nucleic Acid Drug with siRNA Specific Expressed

作　　者：杨磊[1] 曹以诚[1] 高强[1] 葛洪[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广州518640

出　　处：《生物技术通报》2012年第4期181-185,共5页Biotechnology Bulletin

摘　　要：旨在研究靶向于HBsAg的肝脏特异性siRNA,实现核酸药物高靶向性、高特异性基因治疗乙肝。通过重组PCR的方法构建靶向于HBsAg的肝脏特异性表达的siRNA的核酸药物。将目标载体转染HepG2 2.2.15细胞,用ELISA的方法测定转染细胞培养液内HBsAg的含量。PCR鉴定和测序确定重组PAAV-MCS载体PApoE1-ApoE2-hAAT-siHBsAg-U1 3'Box构建成功。重组质粒对HepG2 2.2.15细胞分泌的HBsAg所产生的抑制作用自转染后5 d达到高峰,抑制率达到(30±0.28)%(P<0.01),siRNA特异性表达单元能显著降低HepG2 2.2.15细胞HBV DNA的拷贝数。成功构建的肝脏特异性、siRNA高表达的核酸药物可以有效地抑制HBV基因的表达和复制,为乙肝的进一步治疗研究做了关键性工作。In order to realize gene therapy for hepatitis B with nucleic acid drug high targeting and specificity,the liver-specific siRNA targeting the HBsAg in liver was investigated.The nucleic acid drug targeting the HBsAg in liver with the specific siRNA was constructed by recombinant PCR.The recombinant vector was transfected into HepG2 2.2.15 cells,which can excrete HBsAg continuously.The HBsAg in cell culture medium was assayed through ELISA at various days after transfection.The recombinant plasmid PApoE1-ApoE2-hAAT-siHBsAg-U1 3＇Box was successfully verified by PCR and sequence analysis.The recombinant plasmid inhibited the HBsAg produced by HepG2 2.2.15 cells reaching a peak after 5 days of transfection,which got（30±0.28）%（P 0.01）.The siRNA-specific expression units could reduce the HBV DNA replication significantly.The successfully constructed nucleic acid drug of liver-specific and siRNA＇s high expression could suppress HBV gene expression and replication effectively,which did critical work for further study of the hepatitis B.

关 键 词：RNAI 肝脏特异性siRNA 重组PCR HepG22.2.15细胞 ELISA 

分 类 号：R978.7[医药卫生—药品]