湖北地区119例 HCV 1b 型 NS3丝氨酸蛋白酶区的序列变异研究

目的：分析丙型肝炎病毒(hepatitis C virus, HCV)1b 型非结构蛋白3(non-structure protein 3, NS3)丝氨酸蛋白酶区序列的变异规律及影响意义。方法使用基因型别特异性引物,通过巢式 PCR 的方法扩增119例慢性 HCV 1b 型患者血清中 HCV NS3区。对 PCR 产物进行测序后获得 NS3区核苷酸及氨基酸序列。分析119例 HCV 1b 型 NS3区序列的同源性及种系进化,分析 NS3丝氨酸蛋白酶区的变异情况及重要功能位点的突变情况。结果湖北地区 HCV 1b 型与 HCV 1b 型标准株及亚洲地区序列同源性较高,核苷酸序列同源性为85.94％及87.68％,氨基酸序列同源性可达96.83％及92.39％,而与欧美地区1b 型同源性较低,核苷酸序列同源性均为85.57％,氨基酸序列同源性分别为92.17％及93.38％。进化树分析提示湖北地区序列与中国其他地区序列亲缘性较接近,而与日本、东南亚地区及欧美地区的1b 型亲缘性较远。 NS3丝氨酸蛋白酶区185个氨基酸内有34个位点有氨基酸突变,但其重要的功能区包括催化酶底物特异性结合位点以及 Zn2﹢结合位点在所有序列中均高度保守,无一例发生突变。结论对湖北地区 HCV 1b型 NS3丝氨酸蛋白酶区的变异规律及生物学意义的研究进一步丰富和完善了对 HCV 1b 型基因组变异情况的认识。为了解 HCV1b 型在中国地区的进化过程提供一定理论基础。     

丙型肝炎病毒全长cDNA模板的构建及鉴定

目的 构建具有功能的丙型肝炎病毒(HCV)全长cDNA克隆。方法 应用长模板RTPCR法扩增1份上海地区HCV感染者血清的RNA(基因型为1b)，分段扩增、融合拼接成9．2kb的基因片段，克隆人含有HCV基因两端非编码区序列的载体作为模板。为检测此过程是否发生对特定变异株的选择，分析4个独立克隆的HVRl序列。对原核细胞中表达的HCV核心蛋白、NS3蛋白酶及解旋酶，以蛋白印迹实验确证其免疫反应性。并构建NS3／4A-SEAP表达系统检验NS3的蛋白酶活性。结果 获得丙型肝炎病毒全长cDNA模板。不同克隆间HVR1序列存在较大差异，提示长模板RT-PCR所制备HCVcDNA具有HCV基因准种(quasispecies)的特性。该模板编码基因在原核细胞内得到高效表达，并具有良好的免疫反应性。在NS3／4A-SEAP表达系统内，NS3可切割、释放其下游的SEAP，具有蛋白酶活性。结论 本工作为构建全长功能性HCV cDNA模板及感染性克隆奠定了基础。     

丙型肝炎病毒蛋白酶抑制肽构建及活性的初步鉴定

目的 根据丙型肝炎病毒(HCV)丝氨酸蛋白酶4个多肽底物切点的氨基酸序列特异性,设计1个多位点的竞争性抑制多肽序列.方法 采用中心模板法PCR合成多肽基因;利用原核高效表达载体pBVIL1在大肠杆菌HB101中,表达目的多肽;提取包涵体,用离子交换层析纯化蛋白;在体外添加到蛋白酶和NS5A-B片段构建的反应系统中,用SDS-PAGE鉴定多肽对病毒蛋白酶的抑制活性.结果 正确合成了多肽基因,重组载体pBVIL1/IP在HB101中表达了目的多肽;纯化后,获得电泳纯多肽;体外初步测定证实,随多肽浓度增加,蛋白酶底物降解受到抑制.结论 原核表达的重组抑制多肽,体外对HCV蛋白酶活性具有抑制作用.     

丙型肝炎病毒非结构区NS3基因在大肠埃希菌中的可诱导性表达

目的 构建丙型肝炎病毒（HCV）NS3基因的原核细胞表达载体。实现在大肠埃希菌中的可诱导性表达。方法 应用聚合酶链反应（PCR）技术，以美国HCV-H株全长cDNA质粒为模板，扩增获得NS3基因片段，克隆到原核表达载体pET-30C( )中，构建原核表达载体pET-NS3，转化BL21（DE3）宿主菌，以IPTG诱导，获得NS3蛋白的可诱导性表达，以HCVNS3的单链可变区抗体（ScFv)证实表达的NS3蛋白的特异性，结果 以HCVNS3基因序列特异性引物，PCR扩增获得1893bp的NS3DNA征段，插入pET-30C( )表达载体，转化BL21（DE3）受体菌，经培养，IPTG诱导，获得了重组HCVNS3蛋白的表达，以HCVNS3的ScFv证实了表达的重组蛋白HCVNS3的特异性。结论 以大肠埃希菌表达了HCVNS3的重组蛋白质。     

丙型肝炎病毒丝氨酸蛋白酶基因痘苗病毒表达载体的构建及序列分析

应用反转录巢式PCR扩增出丙型肝炎病毒丝氨酸蛋白酶基因,并克隆到痘苗病毒表达载体pSC65中。载体上设计一测序引物,经序列测定证明,已将543个bp长的丝氨酸蛋白酶基因片段正确插入载体。此结果为丙型肝炎病毒NS3区丝氨酸蛋白酶基因在痘苗病毒中的表达,以及建立HCV特异的CTL靶细胞打下基础。     

2型登革病毒非结构蛋白NS3的表达及其相互作用蛋白的纯化

目的构建2型登革病毒(DENV2)非结构蛋白3(NS3)与亲和标签融合蛋白的表达质粒,串联亲和纯化(TAP)法获得与NS3相互作用的蛋白。方法根据DENV2基因序列,设计引物;以DENV2 c DNA为模板,PCR扩增出NS3基因,经酶切后克隆至含有串联亲和标签(FLAG-StrepⅡ)的哺乳真核表达载体p CI-SF,获得重组表达质粒p CI-NS3-SF;将上述重组质粒通过转染试剂LipofectamineTM2000瞬时转染进入HEK293T细胞,Western blot法验证NS3融合蛋白的表达;通过TAP法分离纯化与NS3相互作用的蛋白。结果成功构建了NS3融合蛋白表达载体,利用TAP系统分离得到与NS3蛋白相互作用的宿主蛋白。结论串联亲和纯化法可以有效的分离与DENV2 NS3相互作用的细胞蛋白。     

HCV NS5A-B片段的表达及在酶活性研究中的应用

目的：利用原核表达载体pBVIL1，表达丙型肝炎病毒非结构蛋白（NS3）丝氨酸蛋白酶催化底物NS5A—B小分子，为进一步研究蛋白酶活性提供方法学。方法：将PCR合成的NS5A—B（2412—2427aa）基因直接插入高效原核表达载体pBVIL1，融合表达NS5A—B片段．包涵体形式的表达产物用8mol／L脲溶解后，采用离子交换和凝胶过滤两步纯化。利用SDS—PAGE、Western blot和ELISA方法，于37℃酶催化条件下，分析NS5A—B底物的降解活性。结果：（1）构建了pBVIL1／NS5A—B重组质粒，鉴定证明NS5A—B基因片段正确地插入表达载体上：融合蛋白在转化质粒的HB101菌中得到高效表达。分离纯化重组蛋白后浓度可达0．73g／L。（2）在NS3蛋白酶作用不同时间后，用SDS—PAGE和Western blot证实，底物带可被明显降解，ELISA分析也证明，NS5A—B具有酶底物活性。结论：含有酶切位点的NS5A—B融合蛋白可被NS3丝氦酸蛋白酶有效地降解，可用作为NS3蛋白酶的底物，可替代全长NS5A—B和化学合成肽用于酶活性和酶阻断剂的研究     

HCV非结构蛋白质5A基因全长和高免疫源区的表达及免疫活性检测

目的探讨HCV非结构蛋白质5A（NS5A）基因全长和高免疫源区的表达并比较其检测灵敏度。方法以含HCV全基因组的质粒pBR^tm／HCV-3011（1b型）为模板，采用PCR方法扩增出NS5A全长基因（以下用NS5AL代替）和高免疫源区基因（以下用NS5AS代替），与pET-32a载体相连接构建重组表达载体pET-NS5AL和pET-NS5AS，分别转化E.coli Rosetta（DE3）pLysS和BL21（DE3）感受态细胞，经异丙幕-β-D-硫代半乳糖苷（IPTG）诱导后，蛋白质印迹法检测其表达，镍螯合（Ni2^＋-NTA）琼脂糖亲和层析柱纯化重组NS5AL和NS5AS蛋白，最后通过ELISA法检测重组蛋白的免疫活性并对其榆测灵敏度进行比较。结果SDS—PAGE鉴定与蛋白质印迹验证结果表明，重组NS5AL和NS5AS蛋白均得到很好地表达；纯化的重组NS5AL和NS5AS蛋白浓度分别为0．146和0．426mg／ml，纯度均〉96％；ELISA检测结果表明，重组NS5AL和NS5AS蛋白均具有较好的免疫活性，且重组NS5AL蛋白的检测灵敏度明显高于NS5AS。结论成功地表达出重组NS5AL和NS5AS蛋白，均具有较高的免疫活性，将有助于提高HCV临床检测的灵敏度。     

活体荧光成像监测HCV NS3/4A蛋白在小鼠体内的瞬时表达

目的建立HCV NS3/4A蛋白酶在小鼠体内可视化表达模型。方法利用水动力转染技术将融合基因NS3/4A-Fluc转染至小鼠肝脏,建立目的基因的瞬时表达模型,通过RT-PCR方法检测目的基因Fluc及NS3/4A的RNA表达水平,以及通过Western blot方法检测目的基因NS3/4A-Fluc的蛋白表达水平。结果建立了通过报告基因Fluc反映HCV NS3/4A蛋白酶的瞬时表达可视化小鼠模型。结论成功建立了可用于评价NS3/4A蛋白酶的可视化小鼠模型。     

HCV NS3蛋白单克隆抗体的制备及其识别区域的分析

目的 制备针对丙型肝炎病毒（HCV）非结构区NS3全长蛋白的单克隆抗体（MAb）,并分析获得的单抗识别表位所在区域,为建立以NS3蛋白为靶位的抗HCV研究提供抗体工具。方法 用原核表达的HCV非结构区NS3全长蛋白作为免疫原,采用小鼠腹股沟皮下NC膜包埋法免疫小鼠,按常规杂交瘤细胞的制备方法,经细胞融合、克隆化制备抗NS3蛋白的MAb。用间接免疫荧光法和Westem blot鉴定其特异性。分别构建NS3丝氨酸蛋白酶（NS3蛋白的N末端1／3）编码基因的真核表达质粒pcDNA3．1（-）-ns3p、NS3解旋酶（NS3蛋白的C末端2/3）编码基因的真核表达质粒pcDNA3．1（-）-ns3A,将其瞬时转染COS-7细胞后,以获得的单克隆抗体作为一抗,通过免疫荧光分析获得单抗识别表位所在的区域。结果 获得了2株抗NS3蛋白的单克隆抗体,这2株MAbs均特异识别NS3蛋白,并确定了它们的结合区域。结论 获得了针对NS3蛋白的单克隆抗体,并对其单抗识别表位所在的区域进行了分析,为下一步进行以NS3蛋白为靶位的抗HCV研究奠定了良好的基础。     

A novel high throughput screening assay for HCV NS3 serine protease inhibitors

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.     

HCV NS3 N末端部分氨基酸缺失对NS3/NS3与NS3/NS4A分子间相互作用的影响

目的旨在弄清ＮＳ３参与分子间相互作用的确切区段，为研究针对ＮＳ３的抗ＨＣＶ寡肽小分子药物的设计提供依据。方法参照ＨＣＶ中国河北株序列设计ＮＳ３引物，将其Ｎ末端的前１５个和前３０个氨基酸分别缺失掉。然后用酵母双杂交系统检测ＮＳ３／ＮＳ３及ＮＳ３／ＮＳ４Ａ分子间相互作用强度在缺失前后的变化，从而判明ＮＳ３Ｎ末端氨基酸在分子间相互作用中的意义。核苷酸序列分析采用ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍ３７３Ａ型自动测序仪。结果ＮＳ３Ｎ末端氨基酸缺失前后，ＮＳ３／ＮＳ３分子间及ＮＳ３／ＮＳ４Ａ分子间相互作用的强度相差有显著性（Ｐ＜０．０１），但缺失１５个氨基酸和缺失３０个氨基酸对上述相互作用强度的影响差异无显著性（Ｐ＞０．０５）。结论ＮＳ３Ｎ末端的１～３０个氨基酸在ＮＳ３／ＮＳ３及ＮＳ３／ＮＳ４Ａ分子间相互作用中有一定意义，其Ｎ末端前１５个氨基酸（ＡＰＩＴＡＹＳＱＱＴＲＧＬＬＧ）对于分子间相互作用更为关键。本研究结果将为抗ＮＳ３丝氨酸蛋白酶活性的寡肽抑制物的研究打下基础，并为抗ＨＣＶ的寡肽小分子药物的设计提供依据     

Internal cleavage of hepatitis C virus NS3 protein is dependent on the activity of NS34A protease

The nonstructural protein NS3 of the hepatitis C virus (HCV) is indispensable for virus replication and a multifunctional enzyme that contains three catalytic activities such as serine protease, helicase, and NTPase. Here, we demonstrated that the internal cleavage of the HCV NS3 protein occurs in various mammalian cells such as HepG2, COS-7, and NIH3T3. As is observed for the internal cleavage mechanism of the NS3 protein of dengue virus 2, the internal processing of HCV NS3 protein was catalyzed by the active NS3 serine protease and NS4A, but not NS3 alone. From the data acquired from extensive site-directed mutagenesis, we observed that the NS3 protein was internally cleaved at two different sites, FCH(1395) ||S(1396)KK and IPT(1428) ||S(1429)GD, within RNA helicase domain. The internal cleavage of NS3 protein by NS34A protease was also confirmed in a different isolate of HCV-1b strain. In addition, in vitro transforming assays demonstrated that the internal cleavage product of NS3, NS3a-1, appeared to have higher oncogenic potential than does intact NS3. Taken together, our results suggest that the internal cleavage of NS3 may be associated with the replication and oncogenesis of HCV.     

A comparative analysis of the substrate permissiveness of HCV and GBV-B NS3/4A proteases reveals genetic evidence for an interaction with NS4B protein during genome replication

The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs containing HCV sequences at the C-terminus of NS4B had a pseudo-lethal replication phenotype. Replication-competent revertants contained second-site substitutions within the NS3 protease or NS4B N-terminus, providing genetic evidence for an essential interaction between NS3 and NS4B during genome replication.     

Hepatitis C virus NS2/3 protease regulates HCV IRES-dependent translation and NS5B RdRp activity

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2/3 protease is the first of two virally encoded proteases required for HCV polyprotein processing. In this report, we investigated the function of NS2/3 protease on HCV replication and translation. Cells transfected with plasmids encoding wild-type or mutant NS2/3 and a dual-luciferase reporter construct containing an HCV internal ribosome entry site (IRES) were used to examine the effect of NS2/3 protease on translation of HCV RNA. Cells transfected with plasmids encoding wild-type or mutant NS2/3, pcDNA-NS5B and a reporter plasmid were used to examine the effect of NS2/3 protease on HCV replication. The results showed that both autocleavage processing and the uncleaved form of NS2/3 protease specifically decrease HCV IRES-directed translation, while the uncleaved form of NS2/3 protease decreases HCV NS5B RdRp activity (replication), indicating that autoregulation by NS2/3 protease of HCV replication and translation may play an important role in persistent HCV infection.     

Hepatitis C virus NS3/4A protein interacts with ATM, impairs DNA repair and enhances sensitivity to ionizing radiation

Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Nonstructural protein 3 (NS3) of HCV possesses serine protease, nucleoside triphosphatase, and helicase activities, while NS4A functions as a cofactor for the NS3 serine protease. Here, we show that HCV NS3/4A interacts with the ATM (ataxia-telangiectasia mutated), a cellular protein essential for cellular response to irradiation. The expression of NS3/4A caused cytoplasmic translocation of either endogenous or exogenous ATM and delayed dephosphorylation of the phosphorylated ATM and gamma-H2AX following ionizing irradiation. As a result, the irradiation-induced gamma-H2AX foci persisted longer in the NS3/4A-expressing cells. Furthermore, these cells showed increased comet tail moment in single-cell electrophoresis assay, indicating increased double-strand DNA breaks. The cells harboring an HCV replicon also exhibited cytoplasmic localization of ATM and increased sensitivity to irradiation. These results demonstrate that NS3/4A impairs the efficiency of DNA repair by interacting with ATM and renders the cells more sensitive to DNA damage. This effect may contribute to HCV oncogenesis.     

Characterization of NS3 protease from an Egyptian HCV genotype 4a isolate

The role of the NS3 protease in HCV replication was demonstrated by the ability of a protease inhibitor cocktail (10 μg/ml) to abolish the induced cytopathic effect in RAW macrophages upon infection with Egyptian sera. The HCV protease gene was amplified from Egyptian sera by nested PCR and cloned downstream of the CMV promotor in a mammalian expression plasmid, which was then used to transform bacteria. Colonies carrying the gene in the correct orientation were subjected to large-scale plasmid purification followed by sequencing. Phylogenetic comparison of the sequence obtained with published sequences from different genotypes confirmed that our sequence belongs to genotype 4a. Of the other genotypes, the most closely related ones were from genotype 1. Multiple alignments of protease peptides showed that the catalytic triads and binding residues for substrate, Zn2+ and the NS4 cofactor are conserved among different isolates, including ours, and confirmed the closer homology between NS3 of genotypes 4 and 1. The HCV-protease-encoding construct was successfully transcribed in both mammalian cells and mice. Mouse antibodies produced against the protease-encoding-construct detected the 18-kDa enzyme in lysates of cells transfected with the construct by Western blotting, and in the media of infected cells by ELISA.