HCV包膜蛋白E2的克隆、表达与检测

构建丙型肝炎HCV包膜蛋白糖蛋白的E2基因原核表达载体,获得大量重组HCVE2蛋白,进行E2蛋白的抗原性及潜在保护作用研究。通过RT-PCR从HCVRNA阳性血清标本中扩增出975bp(383～708)E2基因片段,PCR产物经EcoR I和Sall I双酶切后连接到经同样酶切的PET-41a原核表达载体上,转化到大肠杆菌BL21(DE3)菌株,经Amp筛选,得到阳性重组质粒PET41a-HCVE2菌株,并以IPTG诱导蛋白表达,SDS-PAGE鉴定,表达产物经固定化金属配体亲和层析纯化,用ELLSA方法检测生物学活性。结果表明,构建的HCVE2包膜蛋白基因片段原核表达质粒所表达产物主要以包涵体形式存在,表达的融合蛋白与HCV阳性血清具有较好的反应原性。以HCVE2融合蛋白检测患者阳性血清具有良好的抗原性,有望能提高HCV抗体检测试剂盒的检出率。     

在哺乳动物细胞中稳定表达丙型肝炎病毒E2糖蛋白

利用ＤＮＡ重组技术,将Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因片段插入真核表达载体,然后转染哺乳动物细胞ＮＩＨ３Ｔ３以表达Ｅ２糖蛋白．检测显示来自３月以上培养的细胞克隆中表达产物分子量为７０ｋＤ,经Ｗｅｓｔｅｒｎｂｌｏｔ证实该表达产物能与抗ＨＣＶ阳性血清进行特异性反应．以上表明首次在哺乳动物细胞中成功表达Ⅲ型中国株ＨＣＶ的Ｅ２糖蛋白,并建立相应的稳定表达细胞系．     

Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins

Hepatitis C virus proteins are synthesized as a polyprotein cleaved by a signal peptidase and viral proteases. The behaviour of internal signal sequences at the C-terminus of the transmembrane domains of hepatitis C virus envelope proteins E1 and E2 is essential for the topology of downstream polypeptides. We determined the topology of these transmembrane domains before and after signal sequence cleavage by tagging E1 and E2 with epitopes and by analysing their accessibility in selectively permeabilized cells. We showed that, after cleavage by signal peptidase in the endoplasmic reticulum, the C-terminal orientation of these transmembrane domains changed from luminal to cytosolic. The dynamic behaviour of these transmembrane domains is unique and it is linked to their multifunctionality. By reorienting their C-terminus toward the cytosol and being part of a transmembrane domain, the signal sequences at the C-terminus of E1 and E2 contribute to new functions: (i) membrane anchoring; (ii) E1E2 heterodimerization; and (iii) endoplasmic reticulum retention.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.     

丙型肝炎病毒核心蛋白在大肠杆菌中的表达

目的：建立稳定表达丙型肝炎病毒(HCV)核心蛋白的原核表达系统,获得高产量的纯化核心蛋白。方法：应用多聚酶链反应(PCR),以HCV—H株全长cDNA序列为模板,扩增获得核心区基因片段,克隆入原核表达载体pBVIL1,构建原核表达载体pBVIL1-C,转化HB101宿主菌,通过温度诱导表达核心蛋白。结果：扩增得到目的基因长度为573bp,构建pBVIL1-C表达载体,在HB101宿主菌中通过温度诱导获得稳定表达,表达蛋白占菌体总蛋白含量的21％,Western—Blot检测证实表达产物可与HCV患者阳性血清发生特异性结合反应。结论：HCV核心蛋白可在大肠杆菌中获得高表达并具有良好的反应原性。     

丙型肝炎病毒囊膜蛋白基因DNA疫苗的构建及动物免疫试验

丙型肝炎病毒(Hepatitis C virus,HCV)主要通过输血传播,可以导致多种临床型的肝炎及其它肝脏疾病,包括肝硬化及肝细胞癌[1,2].目前尚无一种有效的抗HCV的措施,这使人们把眼光放到该病的预防上,DNA疫苗的产生为研制丙型肝炎(丙肝)的预防及治疗性疫苗提供了新的思路.     

日本脑炎病毒SA14-14-2 E蛋白结构域Ⅲ的抗原性和免疫原性分析

为了表达日本脑炎病毒囊膜蛋白(E蛋白)结构域DⅢ区,了解其作为亚单位疫苗的可能性,本研究根据SA14-14-2病毒株序列(GenBank Accession No.D90195)设计两条引物,以全长JEV感染性克隆pBR-JTF为模板,通过PCR扩增出JEVE蛋白DⅢ的cDNA片段,构建了原核表达载体pET-JEDⅢ,转化大肠杆菌Rosetta(DE3)进行融合表达。融合蛋白为可溶性表达,表达量约占菌体蛋白的75%。用纯化后蛋白免疫新西兰兔和BALB/C鼠,通过ELISA,Western blotting,噬斑减少实验,及乳鼠攻毒实验验证JEDⅢ的抗原性和免疫原性。Western blotting及ELISA结果表明纯化后的表达产物具有良好的抗原性,纯化的JEDⅢ蛋白免疫新西兰兔,可以获得高达1:7×105滴度的抗JEV特异性抗体;JEDⅢ蛋白免疫BALB/C鼠,可以获得1:8.2×104滴度的抗JEV特异性抗体。并且获得1:256滴度的中和抗体,乳鼠攻毒实验能达到75%的保护效果。以上结果说明本研究表达、纯化的重组JEDⅢ蛋白,免疫小鼠以及兔后,能产生抗JEV的特异性抗体,中和性抗体,能够保护部分乳鼠接受毒...     

河北省丙型肝炎病毒基因分型研究

丙型肝炎病毒 (Hepatitis C virus, HCV)感染是输血后肝炎的主要原因[1],主要通过输血或使用污染的血制品传播[2],且与肝硬化和肝细胞癌的发生有密切关系.     

Expression, purification and characterization of the Hepatitis B virus entire envelope large protein in Pichia pastoris

The current HBsAg vaccine has performed a vital role in preventing the transmission of HBV during the past 20 years. However, a number of individuals still show no response or a low response to the vaccine. In the present study, the HBV envelope large protein gene was cloned into the eukaryotic expression vector pPIC9k and was subsequently expressed in the yeast Pichia pastoris. The HBV large protein (L protein) was produced and secreted into the medium, where some of the L protein formed particles. The soluble L protein and particles were purified by column chromatography and sucrose density gradient centrifugation. Western blot analysis demonstrated that the particle was composed of both HBV L and S protein. To compare the antigenicity of the L protein and HBsAg, rabbits were immunized with the soluble L protein and the commercially available HBV vaccine and the increasing level of antibodies was determined by ELISA. The results showed that the anti-HBsAg antibody, from rabbits injected with the L protein at a dose of 2 and 10microg, was detected on day 14, whereas rabbits vaccinated with 10 and 2microg HBsAg did not develop antibodies until day 21 and 28, respectively. The antibody level in groups inoculated with the L protein was approximately 50% higher than in the group injected with HBsAg using the same dose. Furthermore, 2microg L protein induced a significant and rapid anti-HBsAg antibody response than 10microg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection.     

细胞内表达的小干扰RNA靶向丙肝病毒5′保守区的研究

将丙型肝炎病毒(HCV)基因组的5′非编码区(5′UTR)插入到报告基因绿色荧光蛋白(eGFP)和荧光素酶(luciferase)的上游,并构建基于Ⅲ型启动子的表达载体,这种载体能产生针对HCV 5′UTR的小干扰RNA。然后将含有HCV 5′UTR的eGFP/luciferase和能产生小干扰RNA的质粒共转染入Hela细胞,通过测定细胞发出的荧光和化学发光强弱来观测抑制效果。实验结果表明,与HCV 5′UTR特异性小干扰RNA表达质粒共转染的细胞无论从定性还是从定量上所测得的荧光和化学发光强度都明显低于阴性对照,且细胞密度经核染色与对照组无明显区别。这揭示了小干扰RNA确实能引起HCV特异基因如5′UTR的沉默,且转染进去的小干扰RNA表达质粒对细胞没有毒害作用。这一工作是通过载体直接在细胞内表达小干扰RNA(siRNA)而不是化学合成的,可以使小干扰RNA在细胞内得到稳定表达,因此本研究设计的siRNA表达载体不仅可以有效沉默HCV 5′UTR,而且该系统可以灵敏地筛选更有效的针对HCV的siRNA,因而这一结果为研究利用RNA干扰进行基因治疗HCV感染做了初步探索。     

戊肝与丙肝病毒在献血员人群中感染状况的对比研究

采用市售试剂对武汉地区乡村献血员进行血清抗ＨＥＶ与抗ＨＣＶ检测,两者的阳性率分别为５．７４％及９．３５％。在２８８份有ＡＬＴ记录的单采浆献血员中,有近期ＡＬＴ升高史的献浆者抗ＨＥＶ及抗ＨＣＶ检出率分别为１４．０４％及１４．１８％,均显著高于无近期ＡＬＴ升高史的献浆者。对上述标本同时进行多项血清ＨＢＶ标志检测,抗ＨＥＶ阳性及抗ＨＣＶ阳性组献血员多项ＨＢＶ标志检测结果与相应阴性组比较均未见显著的差别。     

丙型肝炎病毒囊膜蛋白基因DNA疫苗的构建及动物免疫试验

丙型肝炎病毒（Hepatitis Cvirus,HCV）主要通过输血传播,可以导致多种临床型的肝炎及其它肝脏疾病,包括肝硬化及肝细胞癌。目前尚无一种有效的抗HCV的措施,这使人们把眼光放到该病的预防上,DNA疫苗的产生为研制丙型肝炎（丙肝）的预防及治疗性疫苗提供了新的思路。基因免疫是近几年快速发展起来的一种新型方法,它是将带有目的蛋白的编码基因和表达调控序列的质粒DNA导入机体组织后,使目的基因在体内表达,并全方位的刺激机体的免疫系统,     

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis.To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.     

Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice

Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 μg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.     

New monoclonal antibodies against a recombinant second envelope protein of Hepatitis C virus

To study the immunological features of the hepatitis C virus (HCV) envelope protein (E2 protein), new specific monoclonal antibodies (mAbs) were generated. WKA/H rats were immunized with syngeneic cells infected with a vaccinia virus expressing the E2 protein and with soluble E2 protein obtained from Chinese hamster ovary cells with a plasmid-based expression system. By screening hybridoma cells obtained from spleen cells of the immunized rats, three specific mAbs were obtained. One mAb was reactive to a peptide corresponding to the hypervariable region 1 (HVR1) in E2 protein, while the others reacted to regions outside HVR1. The significance of these antibodies for the diagnosis of HCV infection as well as for analysis of the structure of the HCV E2 protein will be discussed.     

Regulation of Hepatitis C virus replication and gene expression by the MAPK-ERK pathway

The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle. In the present study using a Huh7 cell line Con1 with an HCV replicon, we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-α signalling. Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells. It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site. Consistently, a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays. Thus, the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication. In addition, cyclin-dependent kinases (CDKs) downstream of ERK may also be involved in the modulation of HCV replication since roscovitine, an inhibitor of CDKs had a similar effect to that of U0126. Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels. Further, the replication of HCV replicon in Con1 cells was inhibited by IFN-α. The inhibitory effect of IFN-α could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs. It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.     

Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is a causative agent of serious hemorrhagic diseases in humans with high mortality rates. CCHFV glycoprotein Gc plays critical roles in mediating virus-host membrane fusion and has been studied extensively as an immunogen. However, the molecular mechanisms involved in membrane fusion and Gc-specific antibody-antigen interactions remain unresolved largely because structural information of this glycoprotein is missing. We designed a trimeric protein including most of the ectodomain region of Gc from the prototype CCHFV strain, IbAr10200, which enabled the cryo-electron microscopy structure to be solved at a resolution of 2.8 ?. The structure confirms that CCHFV Gc is a class II fusion protein. Unexpectedly, structural comparisons with other solved Gc trimers in the postfusion conformation revealed that CCHFV Gc adopted hybrid architectural features of the fusion loops from hantaviruses and domain III from phenuiviruses, suggesting a complex evolutionary pathway among these bunyaviruses. Antigenic sites on CCHFV Gc that protective neutralizing antibodies target were mapped onto the CCHFV Gc structure, providing valuable information that improved our understanding of potential neutralization mechanisms of various antibodies.     

Determinants of CD81 dimerization and interaction with hepatitis C virus glycoprotein E2

The tetraspanin CD81 plays an essential role in diverse cellular processes. CD81 also acts as an entry receptor for HCV through an interaction between the large extracellular loop (LEL) of CD81 and HCV glycoprotein E2. The E2-CD81 interaction also results in immunomodulatory effects in vitro. In this study, we examined the relationship between the dimeric crystal structure of the CD81 LEL and intact CD81. Using random mutagenesis, amino acids were identified that abolished dimerization of recombinant LEL in regions that were important for intermonomer contacts (F150S and V146E), salt bridge formation (K124T), and intramonomer disulfide bonding (T166I, C157S, and C190R). Two monomeric LEL mutants retained the ability to bind E2, K124T, and V146E, whereas F150S, T166I, C157S, and C190R did not. Introduction of K124T, V146E, and F150S mutations in full-length CD81 did not affect its oligomerization and the effects on E2 binding were less severe than for isolated LEL. These results suggest that the LEL has a more robust structure in the intact tetraspanin with regions outside the LEL contributing to CD81 dimerization.     

河北省丙型肝炎病毒基因分型研究

Using nested RT-PCR and type special primers of HCV, we performed genotype analysis of HCV RNA from five representative cities in Hebei Province. Among 168 samples with HCV RNA, 104 sera was typelb(61.9%), type 2a account for 22.6% (38/168) , and lb/2a mixed-type was 15.5% (26/168) . The results indicate that typelb is the dominance strain in Hebei Province. Compared with type 2a, typelb has important relationship with chronic hepatitis C. This study build up some foundations for diagnosis, treatment and vaccine study of hepatitis C.