Serological study of hepatitis C virus NS5 protein epitopes and their clinical and diagnostic significance

Three peptides corresponding to the 2295-2317 aa NS5 HCV region and individual parts of this region were synthesized. Antigenic properties of these peptides were investigated. The 2295-2317 aa region contains at least two epitopes of different nature. The full-sized peptide is more promising for the diagnostic studies. Optimal conditions for ELISA with this peptide were defined, allowing the maximum complete utilization of the potentialities of both epitopes.     

HCV核心蛋白在大肠杆菌中的表达及免疫学特性研究

目的 研究HCV核心蛋白在大肠杆菌中的非融合表达及重组蛋白的免疫学特性。方法 用DNA重组技术在两种表达质粒（pQE3O和pTrcHisA）、4种宿主菌（DH5a,TOP10,BL21和M15）中表达HCV全长及羟基端部分缺失的核心蛋白（aal～191,aal～154和aal～69）,表达蛋白经SDSF-PAGE检测,免疫印亦分析,亲和层析法纯化后免疫BALB/C小鼠,ELISA法检测免疫小鼠血清中抗HCV抗体水平。结果 HCV核心蛋白C154,C191在pQE30/M15中获得了表达,表达量分别占菌体总蛋白的18.2%和9.3%。免疫印迹分析的结果显示在C154和C191相应位置处出现杂交条带。ELISA结果显示C154和C191诱导小鼠产生了抗HCV抗体。结论 表达的重组蛋白具有抗原特异性和免疫原性。“截断”型HCV核心蛋白的抗原性和免疫原性并未因羧端的部分缺失而减弱。     

丙型肝炎病毒基因组部分E2／NS1蛋白基因在大肠杆菌中的表达

在大肠杆菌中表达了丙型肝炎病毒基因组的部分Ｅ２／ＮＳ１蛋白（氨基酸３６４－５１２）。表达蛋白经ＳＤＳ－ＰＡＧＥ分析，主要表达带的分子量在～４３０００左右。表达蛋白用于检测已用Ｃ３３ｃ及Ｑ２２检测过的血清，发现Ｅ２ＮＳ１引抗体阳性率占Ｃ３３ｃ及Ｑ２２抗体阳性血清的２０％，尚没有发现单独Ｅ２／ＮＳ１抗体阳性的血清。     

Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications

Dengue non-structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The gene encoding NS1 was amplified and cloned in pET28a (+) vector. In order to increase the purity of the recombinant NS1, the transgene was engineered to carry 6× Histidine tags at both N and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli Rosetta-gami cells and the expression conditions viz IPTG concentration, media type, temperature, and harvest time were optimized. The size of the expressed protein was found to be ~45 kDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The NS1 protein was purified under denaturing conditions, to attain the native conformation, NS1 protein was in vitro refolded and dialyzed. The refolded NS1 protein was detected by commercial Immuno chromatographic strip and NS1 specific monoclonal antibodies. IgM antibody capture ELISA was performed using refolded recombinant NS1 protein which recognized the IgM antibodies in dengue-positive samples of acute phase of infection. Our result suggests that rNS1 protein has immense diagnostic potential and can be used in developing point of care diagnostic assays.     

庚型肝炎病毒NS3蛋白在大肠杆菌中的表达

目的　获取具备免疫反应原性的庚型肝炎病毒ＮＳ３ 抗原。方法　从重组质粒Ｉｗｑ２利用ＰＣＲ 反应扩增出ＧＢＶＣ／ＨＧＶ ＮＳ３ 基因片段,克隆至表达载体ｐＰＲＯＥＸ ＨＴａ 后进行核苷酸序列测定,待鉴定正确后经ＩＰＴＧ 诱导重组工程菌,用ＳＤＳＰＡＧＥ 和Ｗｅｓｔｅｒｎ ｂｌｏｔ 对重组蛋白进行鉴定。结果　酶切鉴定和核苷酸序列测定结果表明成功地构建了重组工程菌ｐＨＴＮＳ３／ ＤＨ５α且克隆的基因片段为ＧＢＶＣ／ＨＧＶ ＮＳ３ 基因片段。ＳＤＳＰＡＧＥ 分析诱导表达产物发现在相对分子质量约４３ ８１０处有一条明显的蛋白表达带,占菌体总量的１７ ．７ ％ 。用ＮｉＮＴＡ 亲和层析柱快捷地获得了纯化的重组蛋白。Ｗｅｓｔｅｒｎ ｂｌｏｔ 鉴定发现重组蛋白可与ＧＢＶＣ／ＨＧＶ ＲＮＡ 阳性病人血清发生抗原抗体反应。结论　获得了具备免疫反应原性的ＧＢＶＣ／ＨＧＶ ＮＳ３ 抗原,该重组蛋白可用于ＧＢＶＣ／ＨＧＶ 感染的检测。     

Evaluation of diagnostic assays for hepatitis E virus in outbreak settings

Hepatitis E virus (HEV) is a major cause of hepatitis. We evaluated five HEV antibody diagnostic assays by using outbreak specimens. The Abbott immunoglobulin G (IgG), Genelabs IgG, and Walter Reed Army Institute of Research (WRAIR) IgM assays were about 90% sensitive; the Abbott IgG and WRAIR total Ig and IgM assays were more than 90% specific.     

E2 and NS5: New antigens for detection of hepatitis C virus antibodies

The value of two new hepatitis C virus (HCV) antigens for detection of HCV antibodies was studied. These two recombinant antigens were derived from the nonstructural-5 (NS5) and envelope -2 (E2) region of the HCV genome. In a panel of 33 HCV-RNA positive samples with indeterminate Riba-2 confirmatory test results, 29 samples (88%) showed additional antibody reactivity against E2 and 12 samples (36%) snowed additional reactivity against NS5. Among 39 HCV-RNA negative, Riba-2 indeterminate donor samples, no additional E2 or NS5 reactivity was found in 34 samples (87%); while 5 samples (13%) showed additional reactivity against NS5 and/or E2. E2 reactivity thus resolved the majority of hitherto indeterminate samples. In serial samples from nine posttransfusion hepatitis C patients, NS5 and E2 antibodies did not appear earlier than classical HCV antibodies. However, E2 antibodies eventually appeared in all nine patients. The recombinant E2 might be a candidate antigen for future HCV antibody assays. © 1994 Wiley-Liss, Inc.     

The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein

The hepatitis C virus (HCV) non-structural (NS) 5A protein plays an essential role in the replication of the viral RNA by the membrane-associated replication complex (RC). Recently, a putative NS5A inhibitor, BMS-790052, exhibited the highest potency of any known anti-HCV compound in inhibiting HCV replication in vitro and showed a promising clinical effect in HCV-infected patients. The precise mechanism of action for this new class of potential anti-HCV therapeutics, however, is still unclear. In order to gain further insight into its mode of action, we sought to test the hypothesis that the antiviral effect of BMS-790052 might be mediated by interfering with the functional assembly of the HCV RC. We observed that BMS-790052 indeed altered the subcellular localization and biochemical fractionation of NS5A. Taken together, our data suggest that NS5A inhibitors such as BMS-790052 can suppress viral genome replication by altering the proper localization of NS5A into functional RCs.     

戊型肝炎病毒结构区基因在大肠杆菌中的表达

目的获得可用于研制戊型肝炎疫苗的基因工程抗原。方法用非融合蛋白表达载体ｐＥＴ１１ａ,表达戊型肝炎病毒（ＨＥＶ）第二读码框区（ＯＲＦ２）２２４６６０ａａ段蛋白。结果得到分子量为５００００的表达产物,经Ｗｅｓｔｅｒｎｂｌｏｔ证实,表达产物具有ＨＥＶ特异抗原性。结论该基因工程抗原可能具有制备戊型肝炎疫苗的前景。     

Expression of deletion mutants of the hepatitis B virus protein HBx in E. coli and characterization of their RNA binding activities

The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.     

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。     

Testing the modularity of the N-terminal amphipathic helix conserved in picornavirus 2C proteins and hepatitis C NS5A protein

The N-terminal region of the picornaviral 2C protein is predicted to fold into an amphipathic alpha-helix that is responsible for the protein's association with membranes in the viral RNA replication complex. We have identified a similar sequence in the N-terminal region of NS5A of hepaciviruses that was recently shown to form an amphipathic alpha-helix. The conservation of the N-terminal region in two apparently unrelated proteins of two different RNA virus families suggested that this helix might represent an independent module. To test this hypothesis, we constructed chimeric poliovirus (PV) genomes in which the sequence encoding the N-terminal 2C amphipathic helix was replaced by orthologous sequences from other picornaviral genomes or a similar sequence from NS5A of HCV. Effects of the mutations were assessed by measuring the accumulation of viable virus and viral RNA in HeLa cells after transfection, examining membrane morphology in cells expressing chimeric proteins and by in vitro analysis of RNA translation, protein processing and negative strand RNA synthesis in HeLa cell extracts. The chimeras manifested a wide range of growth and RNA synthesis phenotypes. The results are compatible with our hypothesis, although they demonstrate that helix exchangeability may be restricted due to requirements for interactions with other viral components involved in virus replication.     

A nonisotopic assay method for hepatitis C virus NS5B polymerase

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is responsible for replication of genomic RNA. A novel nonisotopic assay method is described for detecting its enzymatic activity. The 5' end of the in vitro-transcribed template RNA was attached covalently to the surface of a Covalink module using carbodiimide condensation. The RNA strand containing the 3' untranslated region (3' UTR) of HCV at its 3' end was free in the solution. A purified NS5B polymerase and NTPs along with biotin-labeled UTP were added to this module and the polymerization activity could be detected colorimetrically with streptavidin-conjugated alkaline phosphatase.     

Comparison of hepatitis B core antigens derived from human liver or synthesized in Escherichia coli and evaluation of their use in diagnostic assays for anti-HBc IgM

Hepatitis B core antigen (HBcAg) synthesized in Escherichia coli (clone PR1-11) was compared with HBcAg purified from a liver obtained at autopsy of a patient with chronic active hepatitis B. The molecular weight determined by SDS-PAGE with subsequent transfer of the proteins to nitrocellulose paper, incubation with 125I anti-HBc and exposure to X-ray film, was about 21,000 for HBcAg synthesized in E. coli and was slightly lower for the liver-derived HBcAg. In CsCl density gradients the liver-derived HBcAg had one peak at 1.32 g/ml, and the HBcAg synthesized in E. coli had two peaks at 1.34 and 1.30 g/ml. In a comparison of the immunological activity of both antigen preparations in an enzyme immunoassay, the liver-derived HBcAg was detected only up to a dilution of 1:320, whereas the HBcAg synthesized in E. coli was detected in dilutions up to 1:100,000. With both antigens, the same percentage of sera were positive for anti-HBc IgM from patients with acute (100%), convalescent (61%), past (5%) and chronic active (29-37%) hepatitis. These results indicate that tests for anti-HBc IgM performed with HBcAg synthesized in E. coli are at least as sensitive and specific as those using liver-derived HBcAg.     

Mutations in the E2 and NS5A regions in patients infected with hepatitis C virus genotype 1a and their correlation with response to treatment

Heterogeneity of subgenomic regions of hepatitis C virus (HCV) may be associated with response to interferon (IFN) therapy. The amino acid sequences of the PKR/eIF‐2α phosphorylation homology domain (pePHD), IFN sensitivity determining region (ISDR), PKR binding domain (PKRBD), and variable region 3 (V3) were studied in 19 patients before and after 4 weeks of treatment. All patients were infected with HCV genotype 1a and were treated with pegylated‐IFN and ribavirin. Thirteen patients achieved sustained viral response (responders) and six failed to clear viral RNA (nonresponders). The amino acid sequences in the pePHD and ISDR were identical in responders and nonresponders. However, amino acid substitution at position 2252 of PKRBD was significantly different between responders and nonresponders (P = 0.044). A larger number of mutations were observed in the V3 region of responders (P < 0.001). In this region, the amino acid in position 2364 differed between responders and nonresponders (responders: aspartic acid and serine, nonresponders: asparagine, P = 0.018). The amino acid sequences in the regions which were studied did not change after 4 weeks of treatment. It is concluded that the presence of specific amino acids in position 2252 of PKRBD and position 2364 of V3 might be associated with clinical response to IFN. J. Med. Virol. 83:1332–1337, 2011. © 2011 Wiley‐Liss, Inc.     

Expression of capsid proteins and non-structural proteins of waterfowl parvoviruses in Escherichia coli and their use in serological assays.

While there are a number of methods available for detection of antibodies against waterfowl parvoviruses, none is able to differentiate responses against the capsid and non-structural proteins. To enable this, the capsid and non-structural proteins of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) were expressed in Escherichia coli. These proteins were purified and used as antigens in western blotting assays of antibodies against GPV and MDPV. The results showed that 94.7% of the goose and 90.0% of the duck sera collected from the field contained antibodies against GPV or MDPV. Moreover, these sera could be classified into distinct groups based on differences in patterns of western blot reactivity. These different patterns might indicate different stages in infection. Western blotting assays of sera collected from experimentally infected ducks showed that antibodies against the non-structural protein appeared first after infection, followed by antibodies against the capsid protein. It was concluded that the recombinant capsid and non-structural proteins might serve as useful antigens for assays for antibodies against GPV and MDPV. Moreover, because these assays could discriminate between antibodies against the non-structural protein and those against the capsid protein, they may be useful in differentiating vaccinated from infected birds when recombinant capsid protein is used as the vaccine.     

The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage

Viral proteins interact with one another during viral replication, assembly, and maturation. Systematic interaction assays of the hepatitis C virus (HCV) proteins using the yeast two-hybrid method have uncovered a novel interaction between core and NS5A. This interaction was confirmed by in vitro binding assays, and coimmunoprecipitation in mammalian cells. Core and NS5A are also colocalized in COS-7 cells. Interestingly, NS5A is cleaved to give specific-size fragments, when core is coexpressed in mammalian cells. Overexpression of core produced many dying and rounded cells and effects such as DNA laddering and the truncation of poly(ADP-ribose) polymerase 1 (PARP1), both indicators of apoptosis. These observations led us to investigate the link between the induction of apoptosis by core and the cleavage of NS5A. The proteolysis of NS5A and these apoptotic events can be inhibited by caspase inhibitor, Z-VAD, indicating that core induces apoptosis and the cleavage of NS5A by caspases. In cells infected by the HCV, core may provide the intrinsic apoptotic signal, which produces truncated forms of NS5A. The biological function of core-NS5A interaction and the downstream effect of NS5A cleavage are discussed.     

OPG与MBP融合蛋白在大肠杆菌中的表达

目的 克隆人骨保护素（OPG）成熟肽段编码区基因，并在大肠杆菌中表达。方法 采用RT－PCR法，扩增人OPG成熟肽段编码区cDNA，并克隆入原核表达载体pMAL-c2x中，转化BL21（DE3）PlysS大肠杆菌感受态细胞，经0.1mmol/L IPTG诱导后，收集菌体蛋白，进行SDS－PAGE及Western blot鉴定。结果 获得人OPG成熟肽段编码区cDNA，以构建的原核表达载体pMAL-OPG转化菌株后，可表达人OPG和麦芽糖结合蛋白（MBP）的融合蛋白，相对分子质量（M）为85000。表达产物的蛋白量约为菌体总蛋白的13％。Western blot表明，融合蛋白能与抗人OPG多克隆抗体特异性结合。结论 获得人OPG成熟肽全长cDNA，并在大肠杆菌中以OPC－MBP融合蛋白的形式表达。