复制缺损型HBV的构建及其表达反义RNA和显性阴性突变体抗HBV作用的研究

【作者】 孙殿兴；

【导师】 胡大荣； 邬光惠；

【作者基本信息】 第三军医大学 ， 内科学， 2001， 博士

【摘要】 慢性乙型肝炎病毒感染是我国的常见病，迄今尚无满意疗法。基因治疗为乙型肝炎提供了新的治疗策略，已显示出诱人的应用前景。但目前存在的主要问题是载体缺乏导向性及难以向众多的靶细胞导入足量的目的基因。体内有众多的HBV感染细胞，目的基因到达体内如果没有导向性及放大效应，很难获得满意的临床疗效。经我们对HBV基因组的分析，在HBV复制非关键区域改造其基因组结构，使其表达抗HBV产物用于抗乙肝病毒治疗。与其它病毒载体相比，理论上具有明显的优势，它可利用乙肝患者这个天然“包装细胞”，目的基因在慢性HBV感染者体内将有嗜肝导向性及放大效应，在体内长期表达；当HBV复制水平下降时，重组HBV也将随之减少或消失，有可能从根本上克服上述基因载体的缺陷。本课题对HBV基因组作了五种类型改造，使其分别或联合表达反义RNA和显性阴性核心蛋白突变体，并利用EB病毒载体或逆转录病毒载体转导经修饰的HBV全基因组进入细胞，利用表达HBV抗原并分泌HBV颗粒的2.2.15细胞为模型，观察其抗HBV作用及能否利用野生型HBV作为辅助病毒包装并分泌HBV样颗粒。 方 法1．把HBV基因进行如下修饰：①把部分S区基因反向插入HBV基因组， 表达该区域的反义RNA；②把S启动子区基因反向插入HBV基因组， 表达该区域的反义RNA；③在核心蛋白末端与P蛋白重叠区引入突变 点，使其表达核心蛋白与部分P蛋白的融合蛋白；④把核心蛋白末端至 表面抗原之间的区域删除，使其表达核心蛋白与表面膜蛋白的融合蛋 白；⑤在核心蛋白末端与P蛋白重叠区引入突变点，使其表达核心蛋白 与P蛋白的融合蛋白，同时再把部分S区基因反向插入表达反义RNA。L 把上述经修饰的HBV完整复制单元插入潮霉素抗性EB病毒pMEP4载 体，分别构建质粒pMEPSas（表达S区反义KNA人pMEPFas（表达 S启动子区反义RNA人pMEP（P（表达核心蛋白与P蛋白融合蛋白人 pMEPCS（表达核心蛋白与表面膜蛋白融合蛋白人pMEP（PAS（表达 核心蛋白与P蛋白融合蛋白及S区反义RNA人 以空载体pMEP4为对 照，应用脂质体分别转染整合了 HBV全基因的 2．2．15细胞，潮霉素加 压筛选，各组形成的细胞克隆分别混合培养。3．把表达显性阴性核心蛋白突变体的两种突变型HBV全基因插入逆转录 病毒载体GINa，构建质粒pRV（P（表达核心蛋白与P蛋白融合蛋白） 和 pRV（S（表达核心蛋白与表面膜蛋白融合蛋白），以空载体 GINa为 对照，转染包装细胞系并制备重组逆转录病毒，感染二．2刁 细胞。4．连续收集培养上清液，ELISA法检测HBsAg、HBeAg；提取细胞内核 心蛋白颗粒中 HBV DNA，进行 DNA狭缝斑点杂交，以检测对 HBV复 制的抑制作用；提取细胞培养上清液中病毒DNA，分别以各自跨连接 区或跨突变点区引物行PCR检测，以证实有无突变型HBV颗粒的分泌。5．HBV全基因经删除包装信号。区后，分别插入到 G4 18抗性 pCIneo及 潮霉素抗性 pMEP4载体，构建质粒pCI＊HBV和 pMEP刁HBV，分别转 染 HepGZ细胞系，并分别用 G4或潮霉素筛选形成细胞克隆，检测表 达HBSAg及HBCAg较多者作为HBV包装细胞系，转染表达复制缺损 型HBV的质粒以观察包装效果。 会 果1．在 2．2．15细胞内稳定表达多种抗病毒产物，共分为 7组：2．2．15中MEP4、 2．2．15－Sas、2．2．15－Pas、2．2．15－CP、2．2．15（S和 2．2二5－CPAS，以 2．2．15 细胞为对照，计算对HBSAg的平均抑制率分别为：2厂4土3．83％、66．54 士4．45％（p＜0刀1）、55＊ 士3二7％（p＜0刀1）、40刀8士2刀5％（p＜0．01）、52．94 士1．93％（P＜0＊1）和＊．68士5＊7％…＜0＊1）；对HBCAg的平均抑帘率分别 为：4．46士4．250、26．36士l．690（＜0．0）、65．54土3．22％（＜0．01）、52．86 X ）。。 上1．32％（p＜0刀1）、41．60土1石5％（p＜0刀1）和 59二8上2．10％O＜0刀1）；对 HBV复制的抑制率分别为：0％、59．9％、72．8％、82刀％、67二％和 96石％。2．把表达显性阴性核心蛋白突变体的HBV全基因插入逆转录病毒载体 GINa，制备重组逆转录病毒，感染HepGZ细胞后，应用免疫荧光法可 检出HBcAg的表达，感染22三5细胞以观察疗效，分4组：GINa、pRV（P 和 PRV（S组，以 2．2l 5细胞为对照，观察对 HBSAg的平均抑制率分别 为：2．58士3．77％、28．86上4．73％（p＜0＊1）和39．30士7＊4％O＜0＊1）；对 ＊B＜吨的平均抑韦率分另为：3月4土3刀2％、40．38土2．59％（＜0刀1）和 33＊8士4＊7％O＜0．01）；对＊＊V复帘的抑韦率分别为：13．4％、67．2％和 55．8％。3．经对 HBV基因组进行 5种类型改造后，质粒转染 2．2．15细胞，应用 PCR 法检测突变型 HBV的更多还原

【Abstract】 Chronic infection with the hepatitis B virus (I-IBV) is a major problem of public health and a common disease in our country, and currently available therapies have limited efficacy. New antiviral approaches are needed to reduce the risk of developing chronic liver disease and hepatocellular carcinoma. Gene therapy, which has become one of the most attractive antiviral strategies, is bringing a promising light. But HBV gene therapy poses formidable obstacles to gene delivery. The more intractable problem is the development of methods to target the infected tissues or cell type and express efficiently therein. It is impossible for the present vectors to target so many HBV infected liver cells. HBV has many distinct features that make it attractive candidates as vectors for gene therapy of acquired liver diseases. Viral gene expression is directed by hepatocyte-specific promoter-enhancer elements, and, it establishes a stable episomal transcription template. It can selectively target the liver, and it efficiently infects quiescent hepatocytes. If it may express anti-HBV products and meanwhile be packaged, the antiviral function will be amplified in vivo. The fact that a large number of HBV-infected humans appear continuously to harbor the HBV genome and express HBsAg without pathogenic consequences offers a great advantage in terms of long-term maintenance and expression of a recombinant HBV vector bearing a desired gene. A recombinant HBV genome might be attenuated to the extent that the HBV replication level is reduced. So the limitation of other vectors for HBV gene therapy might be overcome. In this study, the HBV genome was manipulated to express antisense RNA or/and dominant negative mutants of HBV core protein. Transducted by EB virus 111 vector or retrovirus vector, the constructs was tested whether it has anti-HBV effects and can still be packaged in helper cell lines. Methods 1. The HBV genome was modified as follows: (1) partial S gene was reversedly recombined back into HBV genome in order to express antisense RNA complementary to S region; (2) S promoter region was reversedly recombined back into HBV genome in order to express antisense RNA complementary to the S promoter region; (3) two nucleotide acids were inserted at BsrBR I site (in the superposition region of core and the P protein) in order to express a fusion protein of core and partial P protein with a stop codon at EcoRI site; (4) BsrBR Ito EcoRI fragment was removed in order to express a fusion protein of core and partial HBsAg; (5) two nucleotide acids were inserted at BsrBR I site as stated above, and then partial S region was reversedly recombined back, which expressed a fusion protein of core and partial P protein combined with antisense RNA complementary to partial S region. 2. The modified HBV genomes in head-to-tail configuration were inserted into the pMEP4 vector with a hygromycin-resistance gene. Accordingly, five constructs were established as follows: (1) pMEP-Sas, which expressed antisense RNA complementary to partial S region of HBV genome; (2) pMEP-Pas, which expressed antisense RNA complementary to S promoter region; (3) pMEP-CP, which expressed a fusion protein of core and partial P protein; (4) pMEP-CS, which expressed a fusion protein of core and HBsAg; (5) pMEP-CPAS, which expressed a fusion protein of core and p更多还原