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目的:乙型肝炎病毒(HBV)的感染,不仅引起急、慢性病毒性肝炎,而且与肝纤维化、肝细胞癌的发生发展密切相关。为深入研究HBV调节表达的机制,我们应用噬菌体展示技术,以HBV核心启动子DNA片段为固相支持物,筛选肝细胞cDNA文库,获得HBV核心启动子的肝细胞结合蛋白-羧肽酶N(CPN)。CPN是一种从多肽和蛋白质C端氨基酸残基分离的血浆金属蛋白酶,他在调节激肽和过敏毒素的生物活性上起关键作用。CPN是分子量280kD的四聚体,包含两个50KD酶性亚单位和两个83kD调节亚单位。核心启动子产生两个3.5kb RNA:前-核心和前基因组RNA。前-核心RNA编码前-核心蛋白和e抗原,前基因组RNA不仅作为mRNA编码核心蛋白和聚合酶蛋白,而且与病毒蛋白一起包埋入核衣壳,作为模板逆转录。前基因组RNA的表达调控在病毒复制周期中起着关键作用。核心启动子分为两部分:基本核心启动子和核心上游调节元件(CURS),其上游为负性调节元件(NER,1616-1621nt)。CPD在体内与HBV核心启动子结合的作用还不清楚,我们分别构建HBV核心启动子及羧肽酶N的重组载体,通过脂质体转染NIH 3T3细胞,研究CPN对核心启动子的调节表达。方法:根据HBV核心启动子及羧肽酶N的序列设计引物,在核心启动子的引物两端引入MluⅠ和NheⅠ的酶切位点,在羧肽酶N的引物两端引入EcoRⅠ和BamHⅠ的酶切位点。用聚合酶链反应(PCR)的方法分别扩增HBV核心启动子和羧肽酶N基因,克隆到pGEM-Teasy载体上。核心启动子经MluⅠ和NheⅠ双酶切回收连接到同样酶切的pCAT载体上,羧肽酶N经EcoRⅠ和BamHⅠ双酶切回收连接到同样酶切的pcDNA3.1(-)质粒上,构建HBV核心启动子的报告载体及羧肽酶N的真核表达载体,脂质体法瞬时转染NIH 3T3细胞。结果:HBV核心启动子和羧肽酶N(CPN)的PCR产物经1％琼脂糖电泳鉴定与预期大小符合,分别为206 bp和580 bp。重组载体经双酶切鉴定后,证明HBV核心启动子的报告载体及羧肽酶N(CPN)的真核表达载体构建成功。脂质体法瞬时转染NIH 3T3细胞48h后,用ELISA法检测β-gal的表达,显示核心启动子在羧肽酶N(CPN)的影响下,其活性有大约5-6倍的增加。通过体内实验证明羧肽酶N(CPN)可以上调HBV核心启动子的表达。结论:HBV核心启动子结合蛋白羧肽酶N(CPN)与HBV核心启动子共转染细胞,明显调节HBV核心启动子的表达,为进一步研究HBV复制的分子生物学机制提供了新的理论基础。
Objective: Hepatitis B virus (HBV) infection not only causes acute and chronic viral hepatitis, but also is closely related to the occurrence and development of liver fibrosis and hepatocellular carcinoma. In order to further study the mechanism of HBV regulation and expression, we used phage display technology to screen the hepatocyte cDNA library by using HBV core promoter DNA fragment as the solid phase support to obtain the hepatocyte-binding protein-carboxypeptidase N CPN). CPN, a plasma metalloprotease isolated from the C-terminal amino acid residues of polypeptides and proteins, plays a key role in regulating the biological activity of kinins and anaphylatoxins. CPN is a tetramer of 280 kD in molecular weight and contains two 50 kD enzymatic subunits and two 83 kD regulatory subunits. The core promoter produces two 3.5 kb RNAs: pre-core and pre-genomic RNA. Pre-core RNA encodes pre-core and e-antigens. Pregenomic RNA not only encodes core and polymerase proteins as mRNA, but also encapsidates with the viral proteins into the nucleocapsid as a template for reverse transcription. Pre-genomic RNA expression regulation plays a key role in the viral replication cycle. The core promoter is divided into two parts: the basic core promoter and the core upstream regulatory element (CURS), upstream of which is a negative regulatory element (NER, 1616-1621nt). The role of CPD binding to HBV core promoter in vivo is unclear. We constructed recombinant vectors of HBV core promoter and carboxypeptidase N, respectively, and transfected NIH 3T3 cells by liposome to study the regulation expression of CPN on core promoter . Methods: According to the sequence of HBV core promoter and carboxypeptidase N, primers were designed. Mlu and Nhe I restriction sites were introduced into both ends of the core promoter. EcoRI and BamH I enzymes were introduced into both ends of carboxypeptidase N Cut the site. HBV core promoter and carboxypeptidase N gene were respectively amplified by polymerase chain reaction (PCR) and cloned into pGEM-Teasy vector. The core promoter was digested by MluI and NheI and ligated into the same pCAT vector. The carboxypeptidase N was digested with EcoRⅠand BamHⅠ and ligated into the same digested pcDNA3.1 (-) plasmid to construct HBV The core promoter reporter vector and carboxypeptidase N eukaryotic expression vector, liposome method transiently transfected NIH 3T3 cells. Results: PCR products of HBV core promoter and carboxypeptidase N (CPN) were identified by 1% agarose electrophoresis and matched the expected size, which were 206 bp and 580 bp, respectively. The recombinant vector was identified by double enzyme digestion, the HBV core promoter reporter vector and carboxypeptidase N (CPN) eukaryotic expression vector was constructed successfully. The NIH 3T3 cells were transiently transfected by liposomes for 48h, and the expression of β-gal was detected by ELISA. The results showed that the activity of core promoter increased about 5-6 fold under the influence of carboxypeptidase N (CPN). In vivo experiments show that carboxypeptidase N (CPN) can up-regulate HBV core promoter expression. CONCLUSION: The co-transfection of HBV core promoter carboxypeptidase N (CPN) and HBV core promoter into cells significantly regulates the expression of HBV core promoter, providing a new theoretical basis for further study on the molecular biological mechanism of HBV replication .