针对HBVx基因siRNA重组腺病毒制备及其对HBVx基因表达抑制.docVIP

针对HBVx基因siRNA重组腺病毒制备及其对HBVx基因表达抑制 　 作者：秦炜炜，刘家云，张瑞，王涛，王凯，孟艳玲，杨安钢 【关键词】 乙型肝炎病毒；,RNA干扰；,腺病毒；,基因治疗 　　Preparation of recombinant adenovirus mediated siRNA targeting HBVx gene and its suppressive effect on HBVx gene expression 　　【Abstract】 AIM: To construct the recombinant adenovirus vectors for siRNA targeting HBVx gene （HBx） and to evaluate the inhibitive effect on the expression of HBx gene in human hepatoma HepG2 cell line. METHODS: The shuttle vector pShuttleHBx was cotransfected into E. coli BJ5183 with adenovirus backbone plasmid. The homologous recombinants were transfected into HEK293 packaging cells and adenovirus was packaged and amplified, followed by infection of the HepG2 cells transfected with HBx gene. The expression levels of HBx were determined by RTPCR and Western Blot after the total RNA and protein were collected. RESULTS: The recombinant adenoviral vectors containing siRNA targeting HBx gene were successfully constructed, which were confirmed by restriction enzyme digestion. RTPCR results demonstrated that the HBx mRNA was markedly decreased and Western Blot results showed that the expression of HBx protein was also significantly reduced in HepG2 cell line. CONCLUSION: Adenovirusbased siRNA targeting HBx gene can inhibit HBx expression with great potency and specificity in vitro. 　　【Keywords】 hepatitis B virus; RNA interference; adenovirus; gene therapy 　　【摘要】 目的：构建针对乙型肝炎病毒x基因（HBx基因）的小干扰RNA (siRNA)重组腺病毒表达载体，并在能表达HBx基因的人肝癌细胞株HepG2中观察其对HBx基因表达的抑制作用. 方法：重组腺病毒穿梭载体pShuttleHBx与骨架载体在大肠杆菌BJ5183中同源重组得到重组腺病毒载体，后者转染HEK293细胞，包装并扩增出病毒颗粒. 用获得的病毒感染能表达HBx基因人肝癌细胞株HepG2，收集细胞总RNA和总蛋白，采用RTPCR和Western Blot方法检测细胞中HBx基因表达的变化. 结果：经限制性内切酶酶切鉴定，证实针对HBx基因的siRNA重组腺病毒载体构建成功. RTPCR和Western Blot方法证实，在HepG2细胞中，HBx基因在mRNA和蛋白水平均有降低. 结论：腺病毒介导的针对HBx基因的siRNA在体外能够高效、特异地抑制HBx基因的表达. 　　【关键词】 乙型肝炎病毒；RNA干扰；腺病毒；基因治疗 　　0　引言 　　乙型肝炎病毒（hepatitis B virus, HBV）基因组中，HBx基因编码的HBX蛋白是HBV编码蛋白中唯一具有多种调控功能的病毒蛋白质［1-2］，可以激活宿主细胞中受损DNA的修复［3-4］，并且同多种肿瘤相关分子有相互作用. 因此认为，HBX与肝细胞肝癌的发生和发展密切相关. RNA干扰(RNA interference, RN