丙型肝炎病毒包膜糖蛋白E2研究进展

丙型肝炎病毒包膜糖蛋白Ｅ２研究进展吴朝栋，陶其敏Ｅ２糖蛋白（ｓｅｃｏｎｄｅｎｖｅｌｏｐａｇｌｙｃｏｐｒｏｔｅｉｎ）被认为是丙肝病毒颗粒的包膜蛋白成分，随着ＨＣＶ的分子生物学研究深入，目前对Ｅ２糖蛋白的衍变、体外表达已较清楚地阐明，对其运用已逐渐开展，...     

包膜糖蛋白序列分析鉴定丙型肝炎病毒宫内感染

目的从分子水平鉴定丙型肝炎病毒（ＨＣＶ）宫内感染的可能性，为确定ＨＣＶ垂直传播提供更直接的依据。方法分别用免疫分析法和逆转录－聚合酶链反应检测婴儿和母亲的血清抗ＨＣＶ和ＨＣＶＲＮＡ，对母婴ＨＣＶＲＮＡ阳性的血清作ＨＣＶ包膜糖蛋白Ｅ２高可变区（Ｅ２ＨＶ）序列分析和比较。结果１１例ＨＣＶ感染的母亲所生的婴儿有６例生后脐血和静脉血抗ＨＣＶ阳性，５例于１～５个月阴转，另１例持续阳性达１３个月；５例于生后ＨＣＶＲＮＡ阳性，３例于１～６个月自然转阴，２例持续阳性分别达９个月和１３个月；对２例持续ＨＣＶＲＮＡ阳性的婴儿和其母亲Ｅ２ＨＶＲ序列分析发现，生后母婴间的氨基酸同源性高达９３．３３％，而２例母亲间同源性６２．２２％。结论丙型肝炎可以发生母婴传播，并可能为宫内感染。     

重组丙型肝炎病毒包膜蛋白2抗体检测的临床意义

我们利用在大肠杆菌中表达的重组E2蛋白进行抗体检测并分析其临床意义。1.材料与方法:（1）材料:收集100份献血员血清标本,及158例丙型肝炎（丙肝）患者、20例乙型肝炎（乙肝）患者、20例非甲非戊型肝炎患者的血清标本。检测抗-E2所用重组E2蛋白（aa385-aa730）在大肠杆菌中表达并经变性条件纯化（表达所用 HCV E2 cDNA来自Ib型 HCV克隆）。（2）方法:建立血清抗-E2 ELISA检测方法:E2包被抗原 0.15μg/孔包被96孔酶标反应板,酶标二抗抗体为山羊抗人IgG-HRP,TMB显色,在AT838酶     

丙型肝炎病毒检测方法的研究进展及其临床意义（综述）

丙型肝炎病毒是单股正链RNA病毒。1991年，国际病毒命名委员会(ICTV)将其归为黄病毒科丙型肝炎病毒属，其基因组含有一个大约9033个核苷酸构成的大开放读码框(ORF)，可编码3010—3033个氨基酸的多蛋白前体，多蛋白N端的1／4依次为核心蛋白(C)和包膜蛋白(E1和E2)等结构蛋白，其余依次为NS2、NS3、NS4和NS5等非结构蛋白，核衣壳蛋白组成核酸，膜蛋白分布于病毒表面，NS3蛋白具有蛋白酶的功能，NS5蛋白为一依赖于RNA的RNA多聚酶。     

人源抗丙型肝炎病毒包膜蛋白E2单链抗体的研究

目的 研制抗丙型肝炎病毒包膜蛋白E2（HCVE2）的人源噬菌体单链抗体，并探讨其临床价值。方法 以重组的HCVE2为固相抗原，利用亲和筛选的原理，从噬菌体抗体库中经过5轮“吸附-洗脱-扩增”的筛选过程及酶联免疫吸附试验（ELISA）和DNA序列分析。获得HCVE2的人源单链抗体；用该抗体对10例石蜡包埋的丙型肝炎患者肝组织进行免疫组织化学鉴定。结果 ELISA结果表明，制备的HCVE2人源单链抗体（吸光度值A450为1.88)能与HCVE2抗原特异性结合，免疫组织化学结果表明，该抗体能够特异性识别丙型肝炎患者肝组织HCVE2抗原，与正常肝组织及乙型肝炎病毒（HBV）抗原均无交叉反应。结论 此法制备的单链抗体亲和性好，特异性强，且制备方法简便，周期短，为HCVE2病原的检测提供了新的有效的试剂。     

丙型肝炎病毒包膜基因对核心基因DNA疫苗免疫应答强度的影响

目的 研究丙型肝炎病毒(HCV)包膜基因E1E2对核心基因C DNA疫苗诱生的免疫应答作用。方法 将包含HCV C或CE1E2基因片段插入真核表达载体pcDNA3中,构建重组质粒pHCV-C或pHCV-CE1E2,分别免疫Balb/c小鼠,每间隔2wk加强免疫1次,同时剪尾取血。ELISA法检测免疫小鼠血清中HCV C特异性抗体的水平。以pHCV-C转染并表达HCcAg的BLAB/c小鼠骨髓瘤Sp4/0细胞为靶细胞,采用~(51)Cr释放试验检测特异性CTL的杀伤作用。结果 两个实验组20只小鼠均产生抗HCV C特异性抗体,当效/靶细胞比例为100:1时,CTL的杀伤率均明显高于对照组(p<0.01);而pHCV-CE1E2与pHCV-C组之间,无论是抗HCV C抗体的滴度还是CTL的杀伤率均无显著性差异(p>0.05)。结论 E1E2基因的加入,并没有增加HCV C基因DNA疫苗诱导的抗HCcAg特异性抗体的滴度和CTL的杀伤作用。     

丙型肝炎病毒与干燥综合征

干燥综合征(Sjoegren syndrome，SS)是一种以外分泌腺尤其是涎腺和泪腺淋巴细胞浸润为特征的自身免疫性疾病，主要表现为持续口干(口干燥症)和眼干(干燥性角结膜炎)，但全身任何系统、器官均可受累。口眼干燥不伴有其他结缔组织病者称原发性SS，伴有其他结缔组织病者称继发性SS。丙型肝炎病毒(hepatitis C virus，HCV)是1989年发现的一种线性、单链RNA病毒，能够导致慢性肝炎、肝硬化，甚至肝细胞癌，并可引起多种免疫介导的肝外表现，如混合性冷球蛋白血症、肾小球肾炎、扁平苔藓等。近年研究发现HCV感染可能与原发性SS有关。     

丙型肝炎病毒分子生物学研究进展

从1975年起已经确认HAV及HBV是导致人类肝炎的主要病原体。但在输血后肝炎中,有多数并非由HAV,HBV也不是由CMV及EBV所引起的。由于该类病原体感染浓度较低,用常规免疫学方法难以确 认,使人们对它得不到认识,所以称之为非甲非乙型肝炎。这一模糊认识持续了十多年之久,至1988-1989年美国Chiron公司集团Choo等从感染的猩猩血中克隆了一种毒株的片段,证明其基因表达产物能与世界各     

丙型肝炎病毒与甲乙型肝炎病毒重叠感染的研究

对485例病毒性肝炎患者进行了抗HCV、抗HAV-IgM、HBV-M检测.各型病毒性肝炎患者中抗HCV阳性率15.05％,慢性肝炎、肝硬变和重型肝炎阳性率高于急性肝炎;抗HCV阳性者中,27.40％有输血或血浆史;57.53％HBV-M阳性,其中HBsAg阳性占54.76％,抗HBc阳性达88.10％;既往有HBV感染者占33.33％.HBV与HCV重叠感染中慢性肝炎占58.06％,IAV与HCV重叠感染以急性肝炎多见(94.44％),HCV与甲乙型肝炎病毒三重感染可加速肝炎重症化的进程。     

丙型肝炎病毒抗体检测的诊断价值

用国产第二代丙型肝炎病毒抗体（抗－ＨＣＶ）检测试剂盒检测了２９６例临床血清标本以及２８例供血者血清标本。并用聚合酶链反应（ＰＣＲ）方法检测了这些标本的丙型肝炎病毒（ＨＣＶ）ＲＮＡ，以了解抗－ＨＣＶ检测对ＨＣＶ感染的诊断价值。同时探讨其在供血员筛选中的应用价值。结果在临床标本中，抗－ＨＣＶ阳性者有８１．１％ＨＣＶＲＮＡ阳性。抗－ＨＣＶ阴性者有２１．７％ＨＣＶＲＮＡ阳性。但是２８例抗－ＨＣＶ阴性的供血者ＨＣＶＲＮＡ全部阴性。提示：抗－ＨＣＶ阳性对于ＨＣＶ感染的诊断价值很高。临床怀疑为ＨＣＶ感染而进行检测的血清标本即使抗－ＨＣＶ阴性，也不能除外ＨＣＶ感染。用抗－ＨＣＶ筛选供血者可能使供血中ＨＣＶＲＮＡ阳性率大大降低。     

Hepatitis B envelope protein mutants in human hepatocellular carcinoma tissues

Hepatitis B virus (HBV) envelope mutants in the region encoding the highly immunogenic major hydrophilic region (MHR) of surface antigen (HBsAg) have been associated with vaccine failure and chronic infection. To determine if these mutants are associated with the development of human hepatocellular carcinoma (HCC), we measured the frequency and nature of such mutants in 23 HBV-associated HCC and various control tissues by performing Southern blot analysis, the polymerase chain reaction (PCR) and direct sequencing. The HBV genome was present mainly in an integrated form and, in most of the samples, the envelope gene was intact. Amino acid substitutions, involving the MHR region in the HCC tissues, were analysed in 11 (61.1%) of 18 patients with HCC. The mutation Gly145Arg, which has been reported to be associated with immunoevasion, was found in seven of the 18 HCC tissues. A significantly higher frequency of mutations was found in HCC tissues (11 of 18) than in the corresponding non-tumorous tissue of the same patients (one of eight), and in samples from patients with acute (one of 19) or chronic (three of 31) HBV infection ( P < 0.001, Fisher's exact test). The accumulation of these envelope mutants in the HCC tissue suggests that such envelope protein mutations may play a role in the process of oncogenesis and that specific vaccines may need to be developed to prevent the occurrence of mutant HBV-associated HCC. Alternatively, the progressive accumulation of mutants in patients with acute hepatitis, chronic hepatitis and HCC may reflect the increased length of duration of HBV infection in these groups of liver lesions.     

Hepatitis C virus E2 envelope protein induces dendritic cell maturation

Maturation is a critical process for dendritic cells (DC) to gain or enhance their functions in antigen presentation and T-cell activation. In this study, we investigated the effect of hepatitis C virus (HCV) envelope protein E2 on DC maturation and related functions. We show that binding of E2 protein to DC leads to a change from immature to mature phenotype as detected by an increased expression of cell surface molecules including CD83, CD80, CD86, CD11c and MHC class II. The E2-matured DC showed higher capacity to stimulate T-cell proliferation and interferon-gamma production and displayed higher levels of interleukin-12 production when compared with immature DC. The induction of DC maturation by E2 is both time- and dose-dependent and can be inhibited by anti-E2 antibodies. In addition, DC matured by E2 showed decreased uptake of bovine serum albumin and latex beads, indicating their decreased activities of endocytosis and phagocytosis upon maturation. Taken together, our results demonstrated that E2 protein is able to induce dendritic cell maturation and suggested that E2 protein may play an important role in regulation of immune responses during HCV infection.     

乙型肝炎病毒囊膜蛋白候选结合蛋白的研究进展

HBV囊膜蛋白基因具有多个可变的起始编码位置,囊膜蛋白可能包含前前-S、前-S1、前-S2以及主蛋白等区域,不同长度的囊膜蛋白组分与肝细胞内蛋白的相互作用机制并不清楚.本文着重探讨了现有的蛋白-蛋白相互作用的研究手段,总结了目前宿主肝细胞可能与HBV囊膜蛋白相互作用的蛋白,但目前获得的大部分结果需进一步验证.     

Hepatitis B virus

Recent developments in molecular biology have advanced our understanding of the pathogenesis of HBV-induced disease. New data derived from the molecular analysis of clinical material have begun to bridge the gap between bench research and the clinical arena. In this review, we consider topics that have relevance to clinical management and that have not been summarized in the recent literature. The recent advances that have been made in the areas of HBV variants,in vitro cell culture systems, and extrahepatic infection are discussed in greater detail.Supported by grants from the American College of Gastroenterology (B.Y.), Gulf Coast Regional Blood Center (C.N., B.Y.), and Schering Corporation (B.Y.).     

乙型肝炎病毒包膜大蛋白检测与病毒复制的相关性研究

目的探讨乙型肝炎病毒外膜大蛋白(LHBs)与乙肝病毒DNA(HBV DNA)和乙型肝炎e抗原(HBeAg)之间的关系,寻找既方便操作又能反映病毒复制的指标,为乙型肝炎的诊断及疗效评估提供科学依据。方法 410例临床标本分别采用荧光定量PCR技术检测HBV DNA载量,LHBs检测用ELISA法,HBeAg采用化学发光法检测。结果不同HBV-M模式的HBV DNA与LHBs检出结果均无显著性差异(P0.05)。LHBs含量与HBV DNA拷贝数呈正相关(r=0.958,P0.001)。在HBV DNA(+)模式群体中LHBs阳性率明显高于HBeAg(+)人群,差异具有统计学意义(P0.001)。结论血清LHBs与HBV DNA复制密切相关,可作为反映HBV复制的指标。LHBs检测方法特异性优于HBeAg检测方法,LHBs在HBV的诊断和疗效评估方面有重要的临床应用价值。     

Hepatitis B virus taxonomy and hepatitis B virus genotypes

Hepatitis B virus (HBV) is a member of the hepadnavirus family. Hepadnaviruses can be found in both mammals (orthohepadnaviruses) and birds (avihepadnaviruses).The genetic variability of HBV is very high. There are eight genotypes of HBV and three clades of HBV isolates from apes that appear to be additional genotypes of HBV. Most genotypes are now divided into subgenotypes with distinct virological and epidemiological properties. In addition, recombination among HBV genotypes increases the variability of HBV. This review summarises current knowledge of the epidemiology of genetic variability in hepadnaviruses and, due to rapid progress in the field,updates several recent reviews on HBV genotypes and subgenotypes.     

包膜蛋白突变对乙型肝炎病毒包装的影响

目的　探讨包膜蛋白突变对HBV包装的影响。方法　构建包膜蛋白突变的全长HBV基因组表达载体:前S1突变HBV表达载体pHBV mS1、Sloop突变HBV表达载体pHBV mS和前S1、S共突变HBV表达载体pHBV mS1S。分别转染HepG2细胞,以野生型HBV质粒 (adwR9 )转染HepG2细胞为对照;pHBV mS1S和pcDNA3分别与adwR9共转染HepG2细胞。ELISA方法检测上清液中和胞内S抗原;荧光定量PCR检测胞内和上清液中病毒量。结果　突变体pHBV mS1、pHBV mS和pHBV mS1S与对照组adwR9中S蛋白表达量和分泌量无明显差别;单独转染的突变体胞内病毒量较adwR9高,尤其以pHBV mS1S明显,而突变体上清中病毒量较对照组adwR9低,尤其以pHBV mS1S明显;pHBV mS1S与adwR9共转染组上清液中病毒量较pcDNA3与adwR9共转染组低。结论包膜蛋白突变对S蛋白表达量和分泌量无影响,但影响病毒包装,使病毒分泌量下降。