The NS3 protein of hepatitis C virus induces caspase-8-mediated apoptosis independent of its protease or helicase activities

Apoptosis has been implicated in the pathogenesis of hepatitis C virus (HCV)-related disease. Here, we show that expression of HCV NS3, or the NS2/NS3 precursor protein, in mammalian cells results in induction of apoptosis and activation of caspases. HCV NS3-induced apoptosis was blocked by a caspase-8, but not a caspase-9-specific inhibitor. HCV NS3 coimmunoprecipitated with caspase-8, but not with other caspases or with FADD. Coexpression of HCV NS3 and caspase-8 resulted in aggregation of the caspase in punctate structures that colocalized with HCV NS3. Cell lines stably expressing low levels HCV NS3 showed increased sensitivity to Fas-induced cell death. Point mutations of NS3 showed that the pro-apoptotic function of the protein is distinct from its protease and helicase activities. These findings suggest that HCV NS3 promotes caspase-8 induced apoptosis at a pathway site distal to FADD, and that flavivirus NS3 may represent a new class of pro-apoptotic proteins.     

Translocase of outer mitochondrial membrane 70 induces interferon response and is impaired by hepatitis C virus NS3

Hepatitis C virus (HCV) elevated expression of the translocase of outer mitochondrial membrane 70 (Tom70). Interestingly, overexpression of Tom70 induces interferon (IFN) synthesis in hepatocytes, and it was impaired by HCV. Here, we addressed the mechanism of this impairment. The HCV NS3/4A protein induced Tom70 expression. The HCV NS3 protein interacted in cells, and cleaved the adapter protein mitochondrial anti-viral signaling (MAVS). Ectopic overexpression of Tom70 could not inhibit this cleavage. As a result, IRF-3 phosphorylation was impaired and IFN-β induction was suppressed. These results indicate that MAVS works upstream of Tom70 and the cleavage of MAVS by HCV NS3 protease suppresses signaling of IFN induction.     

沉默Apaf-1 基因对氧糖剥夺/ 复氧复糖PC12 细胞线粒体凋亡通路的影响

目的:探讨RNA干扰沉默Apaf-1基因对氧糖剥夺/复氧复糖PC12细胞线粒体凋亡通路的影响。方法:PC12细胞随机分为3组:正常组(Control)、模型组(Model)、Apaf-1基因沉默组(Apaf-1-siRNA)。正常组于CO2培养箱内正常培养,其余2组给予氧糖剥夺2 h、复氧复糖24 h处理,Apaf-1-siRNA组于造模前将化学合成的siRNA通过脂质体转染于PC12细胞靶向沉默Apaf-1基因。用荧光标记的siRNA检测Apaf-1转染效率,Western blot检测转染后PC12细胞Apaf-1蛋白表达,CCK-8检测细胞存活率,TUNEL染色检测细胞凋亡指数,流式细胞术检测细胞凋亡率,免疫荧光染色检测Bax/Bcl-2比值,Western blot检测线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达。结果:Apaf-1-siRNA可有效沉默PC12细胞Apaf-1蛋白表达(P<0.05)。与Control组相比,Model组细胞存活率明显降低(P<0.05),细胞凋亡指数和凋亡率显著升高(P<0.05),Bax/Bcl-2比值升高(P<0.05),线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达显著升高(P<0.05);与Model组相比,Apaf-1-siRNA组细胞存活率显著升高(P<0.05),细胞凋亡指数和凋亡率显著降低(P<0.05),Bax/Bcl-2比值降低(P<0.05),Apaf-1、caspase-9、caspase-3蛋白表达均明显降低(P<0.05)。结论:靶向沉默Apaf-1基因可有效降低氧糖剥夺/复氧复糖PC12细胞线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达,抑制细胞凋亡,提高细胞存活率。     

Survivin and heat shock protein 25/27 colocalize with cleaved caspase-3 in surviving reactive astrocytes following excitotoxicity to the immature brain

Following immature excitotoxic brain damage, distinct patterns of caspase activation have been described in neurons and glial cells. Neuronal cells show activation of the mitochondrial apoptosis pathway, caspase-3 cleavage and apoptotic cell death, while reactive astrocytes show caspase-3 cleavage that is not always correlated with enzymatic protease activity and does not generally terminate in cell death. Accordingly, the aim of the present study was to evaluate the astrocytic colocalization of cleaved caspase-3 and several anti-apoptotic proteins of the inhibitor of apoptosis proteins family (IAPs), such as survivin and cellular inhibitor of apoptosis-2 (cIAP-2), and the heat shock proteins (HSPs) family, Hsp25/27 and Hsc70/Hsp70, which can all prevent caspases from cleaving their substrates. At several survival times ranging from 4 h to 14 days after cortical excitotoxic damage induced by N-methyl-d-aspartate (NMDA) injection at postnatal day 9 in rat pups, single and double immunohistochemical techniques were performed in free floating cryostat sections and sections were analyzed by confocal microscopy. Our results show that survivin and Hsp25/27 are primarily expressed in reactive astrocytes of the damaged cortex and the adjacent white matter. In addition, both molecules strongly colocalize with cleaved caspase-3. Survivin is primarily located in the nucleus, like cleaved caspase-3; while Hsp25/27 is cytoplasmic but very frequently found in cells showing nuclear caspase-3. cIAP-2 was mostly found in damaged neurons but also in some glial scar reactive astrocytes and showed fewer correlation with caspase-3. Hsc70/Hsp70 was only expressed in injured neurons and did not correlate with caspase-3. Thus, we conclude that primarily survivin and Hsp25/27 may participate in the inhibition of cleaved caspase-3 in reactive astrocytes and may be involved in protecting astrocytes after injury.     

Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.     

芹菜素诱导人胃癌细胞凋亡作用及机制研究

目的：研究芹菜素（apigenin, API）致人胃癌细胞凋亡作用及其机制。方法：培养人胃癌BGC823细胞株，加入不同浓度的API，孵育48 h。PI染色流式细胞术（FCM）分析测定凋亡率；罗丹明染色FCM分析测定细胞线粒体跨膜电位(Δψm)；Caspase-9分光光度法检测试剂盒测定caspase-9活性；Western印迹检测线粒体凋亡信号转导通路相关蛋白的表达，包括bax，bcl-2，caspase-9和caspase-3。结果： API（20，40和80 μg/mL）作用48 h能呈浓度依赖性地诱导BGC823细胞凋亡。而且，API也能降低BGC823细胞的Δψm，增加caspase-9活性，促进细胞色素c（Cyt c）释放，上调bax，caspase-9和caspase-3蛋白的表达，同时下调bcl-2蛋白表达，且呈剂量依赖性。结论：API通过活化线粒体信号转导途径诱导人胃癌细胞凋亡。     

Hepatitis C virus core, NS3, NS5A, NS5B proteins induce apoptosis in mature dendritic cells

Although reasons for hepatitis C virus (HCV) persistence are still unknown, specific cellular immune responses appear to influence the pathogenesis and outcome of the infection. Apoptosis of cells infected by viruses may appear suicidal to the viruses that induce programmed cell death of its host. However, apoptosis has been suggested to be a response to virus infection as a mean of facilitating virus dissemination. Annexin V-propidium iodide staining and DNA fragmentation, were used to show that expression of the core, NS3, NS5A, or NS5B protein induces apoptosis in mature dendritic cells. In addition, immunoblotting was used to demonstrate that expression level of p21waf1/cip1 protein decreased in cells expressing one of these HCV proteins. No expression of p53 could be detected and expression of Akt was independent of HCV proteins expression. These results suggest that the effect of these HCV proteins on HCV associated pathogenesis may be linked (at least partially) to its ability to modulate apoptosis pathways in mature dendritic cells.     

Expression of HCV structural proteins impairs IFN-mediated antiviral response

Hepatitis C virus (HCV), especially the genotype 1, is naturally resistant to the antiviral effects of interferon-alpha (IFN-alpha). Expression of the whole HCV genome and the NS5A protein has been suggested to interfere with the antiviral activity of IFN-alpha. Here we have analyzed the effect of individual or various combinations of HCV proteins on IFN-alpha-mediated antiviral effect against vesicular stomatitis virus (VSV). When the structural proteins (core-E1-E2) of HCV genotype 1 were expressed in human osteosarcoma cells in a tetracycline-regulated manner, partial VSV resistance to IFN-alpha was established. This was seen as an enhancement of both viral protein synthesis and production of infectious virus. Priming of core-E1-E2-expressing cells with low doses of IFN-gamma (10 IU/ml) partially restored the antiviral activity of IFN-alpha. The core (high-level expression) and NS4B protein expression also showed some rescue of VSV replication. In this model cell system NS3A-NS4A complex and NS5A showed no inhibition of IFN-alpha-induced antiviral activity. Our results indicate that the expression of structural proteins of HCV may impair the antiviral activity of IFNs.     

SREBP-2沉默抑制衣霉素诱导的软骨细胞内质网应激

目的:探究固醇调节元件结合蛋白2(SREBP-2)对衣霉素诱导的软骨细胞内质网应激(ERS)的影响。方法:分离人正常软骨细胞和骨关节炎(OA)软骨细胞培养,衣霉素和SREBP-2 siRNA分别处理正常软骨细胞。24 h后,实时荧光定量PCR检测对微小RNA-185(miR-185)表达的影响,流式细胞术检测对细胞凋亡的影响,Western blot法检测对SREBP-2、CHOP、p-e IF2α和ATF4等ERS相关蛋白,Bcl-2、Bax和caspase-3等凋亡相关蛋白水平的影响;caspase-3活性试剂盒检测对细胞caspase-3活性的影响。结果:与对照组相比,OA组和衣霉素组SREBP-2表达增加,miR-185表达降低(P0.05)。SREBP-2 siRNA转染可明显阻断衣霉素引起的miR-185降低(P0.05)。miR-185过表达能够下调SREBP-2蛋白水平(P0.05)。与对照组相比,OA组和衣霉素组CHOP、pe IF2α和ATF4的蛋白水平明显上调,Bcl-2表达下调,Bax和caspase-3表达增加(P0.05);SREBP-2沉默处理则显著逆转上述蛋白表达(P0.05)。细胞凋亡率与上述细胞凋亡相关蛋白的变化趋势一致(P0.05)。与衣霉素组相比,SREBP-2 siRNA转染明显下调caspase-3活性,miR-185抑制则明显逆转上述作用(P0.05)。结论:SREBP-2沉默可通过上调miR-185抑制衣霉素诱导的软骨细胞ERS和细胞凋亡。     

Rapid apoptosis induced by Shiga toxin in HeLa cells

Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.     

A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells

Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.     

聚合酶链反应诱导丙型肝炎病毒(HCV)蛋白酶活性位点的突变及突变HCVNS3/4a蛋白的表达

目的 以聚合酶链反应（PCR）突变方法诱导丙型肝炎病毒（HCV）蛋白酶活性位点ser1165的突变，获得全长非结构基因3（NS3）／4a的表达与纯化。方法 分别以NS3 N端正向引物与诱变反向引物，诱变正向引物与NS4a C端反向引物获得2个PCR产物，产物纯化后在新的PCR反应体系中加入以上2个PCR产物与NS3 N端正向引物、NS4a C端反向引物。再次PCR扩增突变模板，分别与野生型模板重组入表达载体pET26-Ub，转化大肠杆菌BL21（DE3）pCG1，诱导表达后经菌体裂解、纯清化、硫酸铵沉淀、DEAE－Sepharose、NTA纯化，Western blot分析表达蛋白的特异性及PCR诱导突变使HCV蛋白酶活性位点失活的作用。结果 获得诱导突变的模板，Western blot证实该突变可完全阻断对NS3丝氨酸蛋白酶与NS3螺旋酶间的切割，部分阻断了螺旋酶与NS4a间的切割，纯化后的HCV NS3／4a蛋白在SDS－PAGE胶上显示为双带。结论 PCR突变方法简便、有效，获得丝氨酸蛋白酶失活的NS3蛋白表达，NS3蛋白与NS4a蛋白以复合物形式存在。     

Suppression of interferon-induced antiviral activity in cells expressing hepatitis C virus proteins.

To elucidate the mechanism of the persistent nature of hepatitis C virus (HCV) infection, we examined whether the expression of HCV proteins affect the antiviral activity of interferon (IFN). Antiviral activity of IFN in HepG2 cells expressing all HCV (type 1b) proteins was much lower than vector control (VC) HepG2 cells when encephalomyocarditis virus (EMCV) was used as a challenge virus. Lesser sensitivity to IFN was also observed in cells expressing NS3, NS4, and NS5 and in cells expressing only NS5A. In contrast, HepG2 cells expressing core, E1, E2, NS2, and NS3 proteins were equally sensitive to IFN as VC cells. We then tested the antiviral activity by IFN in two human amnion-derived FL cell lines expressing NS5A from two different clones, one with an intact sequence of IFN sensitivity-determining region (ISDR) and the other with a mutated ISDR sequence. They were almost equally insensitive to IFN treatment when EMCV was challenged. HCV thus has functional protein(s), possibly NS5A, to suppress IFN-induced antiviral activity and plays an important role in virus-cell interaction and regulation of viral replication.     

过表达脑红蛋白抑制双转基因AD小鼠脑中Aβ诱导的凋亡

目的:研究AD双转基因(APPswe/PS1d E9)小鼠侧脑室注射质粒p NGB后,过表达脑红蛋白(neuroglobin,NGB)对Aβ诱导的AD小鼠脑中细胞凋亡的影响及其潜在机制。方法:将24只鉴定后的13月龄AD双转基因阳性小鼠(雌雄各半)随机分为对照组、侧脑室注射生理盐水+pc DNA3.1(1 g/L)组和侧脑室注射pc DNA3.1(1 g/L)+p NGB组。采用免疫组化检测鼠脑Aβ1-42表达情况,TUNEL染色检测脑内细胞的凋亡情况;Western blot检测鼠脑内与凋亡密切相关的cleaved caspase-3、caspase-9以及PI3K、p-Akt、Akt的蛋白水平。结果:与对照组和注射生理盐水组比较,注射p NGB组小鼠脑内Aβ1-42的表达明显受到抑制(P0.01),TUNEL染色阳性细胞数也明显减少(P0.01);过表达NGB能够明显抑制脑组织内cleaved caspase-3和caspase-9的蛋白表达(P0.01),促进Akt磷酸化水平的增强(P0.01)。结论:p NGB过表达能够明显抑制Aβ的生成并抑制Aβ诱导的细胞凋亡,其机制可能与激活PI3K/Akt通路进而抑制与凋亡密切相关的cleaved caspase-3和caspase-9的表达有关。     

光敏化促进姜黄素诱导人胃癌MGC-803细胞凋亡

目的: 探讨光敏化促进姜黄素诱导人胃癌MGC-803细胞凋亡及其机制。方法: 用MTT法检测光敏化姜黄素对胃癌MGC-803细胞株的增殖抑制率,Hoechst 33258荧光染色观察细胞核形态的变化,流式细胞术检测细胞的凋亡率、线粒体膜电位、细胞内活性氧和Ca²⁺;比色法检测caspase-3、8和9酶活性;Western blotting分析细胞色素C、Bcl-2、Bax和热休克蛋白70(HSP70)水平。结果: 单纯姜黄素(5.0μmol/L)对MGC-803细胞增殖抑制率为(29.74±2.30)%,在光学显微镜下可见部分凋亡细胞,凋亡率为(12.54±1.75)%。而光敏化姜黄素组细胞增殖抑制率则为(44.93±3.61)%,在光学显微镜下能见明显细胞核形态改变,染色质凝集,凋亡小体形成,凋亡率为(26.58±2.67)%,细胞周期主要阻滞于G₀/G₁期。光敏化姜黄素显著降低线粒体膜电位,显著增加细胞色素C、细胞内活性氧和Ca²⁺以及caspase-3、8和9酶活性,与单纯姜黄素组比较,差异显著(P<0.01)。Western blotting结果显示光敏化姜黄素同时显著抑制Bcl-2和HSP70蛋白表达水平。结论: 光敏化姜黄素通过Bcl-2和线粒体途径增强其诱导胃癌MGC-803细胞凋亡的作用。     

沉默FoxM1通过促进线粒体释放细胞色素C诱导口腔鳞癌细胞凋亡

目的:研究沉默叉头框蛋白M1 (FoxM1)基因对口腔鳞癌细胞凋亡影响及机制。方法:口腔鳞癌SCC9细胞感染FoxM1-shRNA慢病毒或阴性对照慢病毒,用RT-qPCR和Western blot测定沉默效果。MTT法测定细胞活力变化,平板克隆实验测定细胞克隆形成能力变化,流式细胞术测定细胞凋亡变化,Western blot测定细胞中cleaved caspase-3和cleaved caspase-9蛋白水平变化,JC-1法测定细胞线粒体膜电位变化,Western blot测定细胞线粒体和胞浆中细胞色素C(cytochrome C)蛋白水平的变化。结果:FoxM1-shRNA慢病毒感染成功下调口腔鳞癌细胞中FoxM1的表达(P0.05),阴性对照慢病毒对细胞中FoxM1表达水平没有影响。沉默FoxM1的口腔鳞癌细胞活力降低(P0.05),细胞克隆形成能力也降低(P0.05),细胞凋亡率及cleaved caspase-3和cleaved caspase-9蛋白水平均升高(P0.05),线粒体膜电位降低(P0.05),胞浆中cytochrome C蛋白水平升高(P0.05),线粒体中cytochrome C蛋白水平降低(P0.05)。结论:沉默FoxM1可以通过降低口腔鳞癌细胞线粒体膜电位、促进线粒体释放cytochrome C而诱导细胞凋亡。     

Endocytic Rab proteins are required for hepatitis C virus replication complex formation

During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.     

丙型肝炎病毒非结构蛋白NS4B肝细胞结合蛋白基因的筛选与克隆

目的筛选并克隆人肝细胞cDNA文库中与丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)相互作用蛋白的基因,明确其具体作用机制。方法应用酵母双杂交系统3,将多聚酶链反应(PCR)法扩增的HCVNS4B基因连接入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基和Xα半乳糖(Xαgal)上进行双重筛选阳性菌落,提取阳性酵母菌落的质粒转化大肠埃希菌,接种在氨苄西林LB平板上,选择生长菌落,提取质粒酶切鉴定,测序并在GenBank中进行生物信息学分析。结果成功克隆出HCVNS4B基因并在酵母细胞中表达,与肝文库配合后选出既能在4缺(SD/TrpLeuAdeHis)培养基又能在铺有Xαgal的4缺培养基上生长,并变成蓝色的真阳性菌落5个,序列分析显示,筛选到的肝细胞蛋白编码基因参与细胞代谢、生物氧化、生长调节等多种生物学过程。结论成功克隆出HCVNS4B蛋白与肝细胞相互作用蛋白,为进一步研究NS4B蛋白的功能,阐明HCV致病的分子生物学机制提供了新线索。