绿色荧光蛋白与 HCV 核心蛋白的融合及在大肠杆菌中的高效表达

利用基因工程重组技术获得了绿色荧光蛋白（ｇｆｐ）基因与ＨＣＶ核心蛋白基因的嵌合体,并在大肠杆菌中高效表达了４８ｋＤａ的融合蛋白,经Ｄｏｔ－ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ ｂｌｏｔ免疫活性分析证实,融合蛋白仍具有ｃｏｒｅ抗原的三个免疫活性部位,同时用荧光显微镜观察并用荧光光度计测定了大肠直菌表达的融合蛋白的荧光光谱,结果证实,我们在大肠杆菌中表达的ＧＦＰ－ｃｏｒｅ融合蛋白既能发射易于检测的绿色荧光,又具     

丙型肝炎病毒核心蛋白抑制乙型肝炎病毒转录的分子机理

为研究丙型肝炎病毒 (HCV)核心蛋白抑制乙型肝炎病毒 (HBV)表达的分子机理 ,从HCV和HBV共感染中国病人血清中分离了 5个含HCV 5′非编码区 ( 5′NCR)和核心区 (CR)cDNA序列的克隆 .序列比较和系统发育分析显示 :从共感染病例中分离的HCV序列与其它已发表的从单独HCV感染病例中分离的序列没有显著差异 .共转染实验证实采用从其中 1例共感染病人中分离的核心蛋白cDNA基因所表达的核心蛋白 ,可抑制HBV表面抗原 (HBsAg)和HBVe抗原 (HBeAg)的表达 ,缺失突变分析说明HCV核心蛋白的C端疏水区对这种抑制作用是必需的 ,报道基因产物分析进一步说明了HBVC启动子和增强子Ⅱ是HCV核心蛋白的效应靶序列之一 ,而HCV核心蛋白在肝细胞和非肝细胞中均对HBV和其他细胞和病毒的基因的转录起负调节作用 .因此 ,认为HCV核心蛋白是一种多功能的负调节因子 ,与HCV和HBV以及HCV与细胞之间的相互作用密切相关     

乙肝病毒增强子对其基因表达的调控

本文构字线性化的含有从核心抗原启动子Ｃｐ起始的ＨＢＶ完整转录单元的基因组隆,以此为模型,通过增强子Ｉ和增强子ＩＩ的缺失或点突变分析,研究了ＥＮＩ和ＥＮＩＩ对ＨＢＶ基因组基因宾调控。结果用ＥＮＩＩ对Ｓ基因表达均有增强作用,且相互协同。     

基因工程表达的乙型肝炎病毒核心抗原转变为e抗原的研究

乙型肝炎病毒核心抗原及e抗原均为乙型肝炎病毒核衣壳的重要组成部分。自从发现病毒核心颗粒中存在着e抗原之后,它们之间的关系一直是人们研究的课题。有些报道认为,e抗原是核心抗原的组成成份。有人通过实验发现核心抗原和e抗原的氨基酸序列极其     

乙肝病毒preS抗原决定簇与核心抗原的融合表达

将乙肝病毒表面抗原的ｐｒｅＳ抗原决定簇片段与核心抗原进行融合,分别构建了在核心抗原中间对应第７５～８３位氨基酸之间的融合及在核心抗原羧端对应第１５６位氨基酸处的融合,并在ｔａｃ启动子的控制下于大肠杆菌中表达。表达产物经ＥＬＩＳＡ检测和ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ分析,表明融合蛋白均被表达,其单体分子量大小与推算值一致．电镜观察和ＣｓＣｌ密度梯度超离心测定都表明融合蛋白能形成颗粒,其密度略小于天然的ＨＢｃ颗粒。初步纯化的融合蛋白免疫Ｂａｌｂ／ｃ小鼠;能产生高滴度的抗－ｐｒｅＳ１抗体,表明ＰｒｅＳｌ（２１～４７）在核心抗原ｅｌｌｏｏｐ区的融合能大大提高其免疫原性．     

丙型肝炎病毒核心蛋白结构及功能研究进展

大量研究结果表明丙型肝炎病毒（ＨＣＶ）核心蛋白是多功能蛋白质,它可能与ＨＣＶ致病（癌）有密切关系。本文简述近年来关于核心蛋白结构与功能的最新进展。并初步探讨了ＨＣＶ核心蛋白可能的致病机制。     

乙型肝炎病毒蛋白和绿色荧光蛋白的共表达研究

目的：构建增强型绿色荧光蛋白（EGFP）标记的乙型肝炎病毒（HBV）真核表达载体,并研究其在真核细胞和小鼠体内的共表达。方法：以质粒pBR322-HBVadr2．0和pCX-EGFP为基础,构建含有双拷贝HBV全基因组DNA和EGFP基因的真核表达载体pCX-EGFP-HBVadr2．0,分别转染真核细胞和小鼠肝组织,建立体外、体内表达系统,研究GFP和HBV基因的表达。结果：构建了真核表达载体pCX-EGFP-HBVadr2．0,EGFP和HBV病毒蛋白在体内和体外均可表达。结论：构建的pCX-EGFP-HBVadr2．0真核表达载体可以GFP作为HBV存在与否的报告基因,提高了培育检测转基因小鼠的效率,为转基因小鼠的制备及后续研究奠定了基础。     

改变乙型肝炎病毒核心抗原基因3′端序列对其在原核细胞中表达的影响

乙型肝炎病毒(HBV)的核心抗原基因(C基因)编码185个氨基酸残基,在原核细胞或痘苗病毒系统中能表达并装配成27nm大小的核心抗原(HBcAg)多聚体颗粒。已证实HBV C基因3′端编码近40个氨基酸的碱基序列,不是表达形成HBcAg颗粒所必需的。用外源基因替换这部分序列,已表达出表面带有外源基因产物的杂合颗粒,它具有很好的免疫原性,成为新型的基因工程多决定簇颗粒载体疫苗。但我们的实验中发现,用另外的外源基因替换3′端序列能显著影响HBV C基因在大肠杆菌中的表达,不同组成的外源基因其影响程度有所不同。     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

乙型肝炎病毒多聚酶末端蛋白的结构和功能研究

乙肝病毒蛋白结构和功能是当前研究乙肝病毒的热点之一。HBV多聚酶的末端蛋白在病毒复制过程中起重要作用,主要包括前基因组RNA包装和DNA合成的蛋白引发等,并可抑制细胞对干扰素的反应。本文综述了乙肝病毒多聚酶末端蛋白的结构和功能,还比较了乙肝病毒与逆转录病毒多聚酶结构和功能的异同。     

乙型肝炎病毒核心蛋白作为表位疫苗载体的应用

乙型肝炎病毒核心蛋白(Hepatitis B viruscore protein,HBc)可以形成二十面体对称的颗粒样结构,由于其N端、C端和主要免疫显性区域(Major immunodominant region,MIR)允许一定程度的缺失和外源插入,并且能够将外源序列重复且高密度地暴露在颗粒的表面,诱发强烈的外源序列特异的体液和细胞免疫反应,从上世纪80年代中期就开始被运用于表位疫苗的研究。以下主要从影响HBc作为表位疫苗载体的因素,包括HBc长度、外源插入位点和表位序列的性质等来介绍HBc作为表位疫苗载体的应用。     

猪丙型肝炎病毒核心蛋白结合蛋白6同源基因的克隆化研究

利用不同种属动物之间重要基因序列高度同源的理论,应用分子生物学与生物信息学技术和方法。克隆猪丙型肝炎病毒(HCY)核心蛋白结合蛋白6(HCBP6)的同源基因。首先应用酵母双杂交技术,以表达HCV核心蛋白的表达栽体作为曾饵,对于百万级的肝细胞cDNA文库酵母进行配合、筛选,首先获得人HCBP6的全长鳊码基因。然后应用美国国立生物工程中心(NCBI)建立的核苷酸序列数据库(CenBank)的同源基因的检索,搜索与之同源的来源于猪的表达序列标签(EST)。然后根据基因同泺性的原则,确定猪HCBP6的同源基因。获得了与HCC核心蛋白结合蛋白的36个基因片段,其中之一命名为HCBP6。根据基因同源性搜索,获得了来源于猪的EST基因序列片段。最终确立了猪HCBP6的同源基因。利用不同物种之间基因同泺性的原理、NCBI数据库GenBank同源基因的搜索,获得了猪HCBP6同源基因。生物信息学技术在后基因组时代具有重要地位和作用。     

Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication

Japanese encephalitis virus (JEV) core protein is detected not only in the cytoplasm but also in the nucleoli of infected cells. We previously showed that a mutant JEV lacking the nucleolar localization of the core protein impaired viral replication in mammalian cells. In this study, we identified a nucleolar phosphoprotein B23 as a protein binding with the core protein of JEV but not with that of dengue virus. The region binding with JEV core protein was mapped to amino acid residues 38 to 77 of B23. Upon JEV infection, some fraction of B23 was translocated from the nucleoli to the cytoplasm, and cytoplasmic B23 was colocalized with the core protein of wild-type JEV but not with that of the mutant JEV. Furthermore, overexpression of dominant negatives of B23 reduced JEV replication. These results suggest that B23 plays an important role in the intracellular localization of the core protein and replication of JEV.     

乙型肝炎病毒X蛋白对细胞凋亡的影响存在剂量依赖性

乙型肝炎病毒(HBV)X蛋白(HBx)与HBV相关肝细胞肝癌(HCC)的发生和发展密切相关.深入研究HBx在HCC形成中的作用将为探索HBV致癌机制提供重要依据.HBx是多功能蛋白,其对细胞凋亡的影响至今仍存在分歧.许多研究表明,HBx既有促进细胞凋亡又有抑制细胞凋亡的功能,但原因不清楚.本研究中,将表达HBx的质粒短...     

Hepatitis B virus core protein interacts with the C-terminal region of actin-binding protein

Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes. There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis. To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis. A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified. This interaction was further confirmed both in vitro and in vivo. In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein. Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278. The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs. The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed.