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1.
Functional Role of Hepatitis C Virus Chimeric Glycoproteins in the Infectivity of Pseudotyped Virus 总被引:20,自引:9,他引:11
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The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5°C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies. 相似文献
2.
Although significant advances have been made on the studies of HCV glycoproteins (E1 and E2) recently, the role of the peptides preceding each glycoprotein remains unclear. We expressed E1 and E2 using two individual plasmids to form HCV pseudoparticles (HCVpp) in order to characterize the peptides preceding E1 and E2.Our data show that 14 amino acids from the HCV core and 12 amino acids from the E1 C-terminus are required for E1 and E2 function, respectively. The lack of a long enough peptide preceding E1 or E2 will abolish HCVpp infectivity, and the presence of fewer than 14 amino acids ahead of E1 and 12 amino acids ahead of E2 may alter their glycosylation. Furthermore, the peptides preceding E1 and E2 may be interchanged or may be replaced by those from genotype 2a. Our findings may contribute to the future development of new anti-HCV drugs. 相似文献
3.
In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 (1a), Hebei (1b) and JFH1 (2a) strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay(IFA) and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV E1-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid (pHR'CMV delta R8.2)and a self-inactivated (SIN) transfer plasmid (pCS-CG) containing a reporter EGFP gene to produce infectious HCV pseudo-particles(pp). Flow cytometry assays showed that the HCVpp could infect Huh7 and Huh7-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25 ng/mL p24 or 10(4)-10(5) TU (transducing unit)/ ml. An in vitro HCV neutralizing assays based on HCVpp (1a, 1b, 2a) were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp (1a, 1b, 2a subtype) were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection (host tropisms, receptor binding, membrane fusion, et al. ) or screening anti-HCV drugs (such as inhibitor to virus entry). This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates. 相似文献
4.
Herrera AM Musacchio A Fernández JR Duarte CA 《Biochemical and biophysical research communications》2000,273(2):557-559
The HIV-1 gp120 gene with natural signal sequence expressed in eukaryotic expression systems showed extremely low levels of synthesis and secretion. Several expression systems have been used to improve the secretion levels of gp 120. In mammalian cells, the efficient expression of gp120 fused to t-PA signal peptide has been previously reported. Here, the effects of t-PA and EPO signal peptides were compared as secretion sequences for expression of gp120 in COS-7 cells. The EPO's signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results indicated that higher amounts of secreted gp 120 were obtained when vectors containing EPO signal peptide were used. 相似文献
5.
口服型HCV融合抗原DNA疫苗在小鼠诱导免疫应答 总被引:1,自引:0,他引:1
将编码一个外源信号肽、一个通用型辅助性T淋巴细胞抗原表位和HCV核心 包膜蛋白E2融合抗原基因的真核表达质粒pST CE2t(DNA疫苗 )转化到减毒鼠伤寒沙门菌SL72 0 7.将该重组菌口服接种BALB c小鼠 3次 .小鼠的抗HCV核心和E2抗体阳转率分别达 6 0 %和 70 % .体外以重组HCV核心或E2抗原刺激小鼠脾细胞 ,均使之发生明显的增殖反应 ,且小鼠脾细胞能有效杀伤表达HCV核心抗原的同系骨髓瘤细胞SP2 0 .这为研制高效免疫、成本低廉、接种方便的HCV疫苗提供了一个新的可行途径 相似文献
6.
S.N. Beljelarskaya O.V. Orlova V.L. Drutsa V.A. Orlov A.V. Timohova N.N. Koroleva V.I. Popenko A.V. Ivanov P.V. Spirin V.S. Prassolov P.M. Rubtsov S.N. Kochetkov 《Biochemistry and Biophysics Reports》2016
Hepatitis C virus (HCV) is characterized by considerable genetic variability and, as a consequence, it has 6 genotypes and multitude of subtypes. HCV envelope glycoproteins are involved in the virion formation; the correct folding of these proteins plays the key role in virus infectivity. Glycosylation at certain sites of different genotypes HCV glycoproteins shows substantial differences in functions of the individual glycans (Goffard et al., 2005; Helle et al., 2010) [1], [2]. In this study, differential glycosylation sites of HCV genotype 1b envelope proteins in insect and mammalian cells was demonstrated. We showed that part of glycosylation sites was important for folding of the proteins involved in the formation of viral particles. Point mutations were introduced in the protein N-glycosylation sites of HCV (genotype 1b) and the mutant proteins were analyzed using baculovirus expression system in mammalian and insect cells. Our data showed that, in contrast to HCV 1a and 2a, the folding of HCV 1b envelope proteins E2 (sites N1, N2, N10) and E1 (sites N1, N5) was disrupted, however that did not prevent the formation of virus-like particles (VLP) with misfolded glycoproteins having densities typical for HCV particles containing RNA fragments. Experimental data are supported by mathematical modeling of the structure of E1 mutant variants. 相似文献
7.
Functional Analysis of Cell Surface-Expressed Hepatitis C Virus E2 Glycoprotein 总被引:5,自引:0,他引:5
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Mike Flint Joanne M. Thomas Catherine M. Maidens Christine Shotton Shoshana Levy Wendy S. Barclay Jane A. McKeating 《Journal of virology》1999,73(8):6782-6790
Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to "native" E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry. 相似文献
8.
Flt3配体增强HCV核心-包膜E2融合基因DNA疫苗诱导的细胞免疫应答 总被引:3,自引:0,他引:3
建立一种可高效诱导细胞免疫应答 ,对丙型肝炎病毒 (HCV)感染可能起预防和治疗作用的DNA疫苗。将小鼠Flt3配体 (FL)信号肽和胞外段cDNA插入结构优化的HCV核心 包膜E2融合抗原DNA疫苗pST CE2t,构建成pST CE2t FL。将pST CE2t FL转染COS7细胞 ,Westernblot和ELISA检测表明该重组质粒能表达HCV核心 包膜E2融合抗原和可溶性小鼠FL。分别将pST CE2t、pST CE2t FL和空载体pCI neo肌肉注射接种BALB c小鼠 ,检测小鼠的体液和细胞免疫应答。结果表明两种DNA结构均能在小鼠体内诱生细胞和体液免疫应答 ,但pST CE2t诱导的体液免疫应答强于pST CE2t FL ,而后者诱导的细胞免疫应答明显强于前者。FL能明显增强HCV核心 包膜E2融合抗原DNA疫苗诱导的细胞免疫应答 ,对于发展HCV预防和治疗性疫苗有潜在的应用价值。 相似文献
9.
生长激素信号肽可诱导重组蛋白外分泌表达 总被引:2,自引:0,他引:2
重组蛋白质的表达是生物医药开发、基因功能和作用机理研究中关键技术环节.虽然细菌表达体系由于表达量大、经济等而被广泛采用,但由于其不能提供许多蛋白质必需的翻译后修饰如糖基化等,所表达的蛋白又多以不可溶包涵体形式存在,变性复性过程复杂,产率低,因此真核细胞表达体系如CHO、COS等成为活性要求高的蛋白质表达的首选[1]. 相似文献
10.
Coexpression of hepatitis C virus E1 and E2 chimeric envelope glycoproteins displays separable ligand sensitivity and increases pseudotype infectious titer 总被引:1,自引:0,他引:1
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We have previously reported that a pseudotype virus generated by reconstitution of hepatitis C virus (HCV) chimeric envelope glycoprotein E1-G or E2-G on the surface of a temperature-sensitive mutant of vesicular stomatitis virus (VSVts045) interacts independently with mammalian cells to initiate infection. Here, we examined whether coexpression of both of the envelope glycoproteins on pseudotype particles would augment virus infectivity and/or alter the functional properties of the individual subunits. Stable transfectants of baby hamster kidney (BHK) epithelial cells expressing either one or both of the chimeric envelope glycoproteins of HCV on the cell surface were generated. The infectious titer of the VSV pseudotype, derived from a stable cell line incorporating both of the chimeric glycoproteins of HCV, was approximately 4- to 5-fold higher than that of a pseudotype bearing E1-G alone or approximately 25- to 30-fold higher than that of E2-G alone when assayed with a number of mammalian cell lines. Further studies suggested that that the E1-G/E2-G or E2-G pseudotype was more sensitive to the inhibitory effect of heparin than the E1-G pseudotype. Treatment of the E1-G/E2-G pseudotype with a negatively charged sulfated sialyl lipid (NMSO3) displayed a approximately 4-fold-higher sensitivity to neutralization than pseudotypes with either of the two individual glycoproteins. In contrast, VSVts045, used as a backbone for the generation of pseudotypes, displayed at least 20-fold-higher sensitivity to NMSO3-mediated inhibition of virus plaque formation. The effect of low-density lipoprotein on the E1-G pseudotype was greater than that apparent for the E1-G/E2-G pseudotype. The treatment of cells with monoclonal antibodies to CD81 displayed an inhibitory effect upon the pseudotype with E1-G/E2-G or with E2-G alone. Taken together, our results indicate that the HCV E1 and E2 glycoproteins have separable functional properties and that the presence of these two envelope glycoproteins on VSV/HCV pseudotype particles increases infectious titer. 相似文献
11.
HCVisthemajorcauseofposttransfusionnonA,nonBhepatitis[1].About50%oftheinfectionswilldevelopintochronichepatitisandamongthemabout20%willresultinlivercirrhosisandhepatocellularcarcinoma[2].BecausethetiterofHCVparticleinpatient’sbloodisextremelylow,andthereisno… 相似文献
12.
Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The
variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the
antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the
different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9
B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3.
This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of
CEMP were characterized by HCV infected patients’ sera, and found that the reactivity frequency reached 75%. The cross reactivity
of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate,
plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of
rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings
indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV
epitopes, and would be of the value as a candidate for the development of HCV vaccines. 相似文献
13.
Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes 总被引:5,自引:0,他引:5
Hepatitis C(HCV) genome is highly variable,particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene.The variability of HCV genome has been a major obstacle for de-veloping HCV vaccines.Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes,we synthesized an minigene of HCV-derived multi-epitope peptide an-tigen(CMEP) ,which contains 9 B-cell HVR1 mimotopes in E2,2 conserved CTL epitopes in C,1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3.This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP.The immunogenic properties of CEMP were characterized by HCV infected patients' sera,and found that the reactivity frequency reached 75%.The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%.Meanwhile,we constructed an HCV DNA vaccine candidate,plasmid pVAX1.0-st-CMEP carrying the recombinant gene(st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene.Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody,which was of the same cross reactivity as the fusion protein GST-CMEP.Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,and would be of the value as a candidate for the development of HCV vaccines. 相似文献
14.
Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells 总被引:4,自引:0,他引:4
Pertussis toxin (PT) comprises an active subunit (S1), which ADP-ribosylates the alpha subunit of several mammalian G proteins, and the B oligomer (S2–S5), which binds glycoconjugate receptors on cells. In a previous report, expression of S1 in Cos cells resulted in no observable cytotoxicity, and it was hypothesized that either S1 failed to locate its target proteins or the B oligomer was also necessary for cytotoxicity. To address this, we stably transfected S1 with and without a signal peptide into mammalian cells. Immunofluorescence analysis confirmed the function of the signal peptide. Surprisingly, we found that S1 was active in both transfectants, as determined by clustering of transfected Chinese hamster ovary (CHO) cells and ADP-ribosylation of G proteins. Constructs with a cysteine-to-serine change at residue 201 or a truncated S1 (residues 1–181) were also active when transfected into cells. Constructs with an inactive mutant S1 had no activity, confirming that the observed results were due to the activity of the toxin subunit. We conclude that S1 is active when expressed in mammalian cells without the B oligomer, that secretion into the endoplasmic reticulum does not prevent this activity and that the C-terminal portion of S1 is not required for its activity in cells. 相似文献
15.
Sulfated homologues of heparin inhibit hepatitis C virus entry into mammalian cells 总被引:1,自引:0,他引:1
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The mechanism of entry of hepatitis C virus (HCV) through interactions between the envelope glycoproteins and specific cell surface receptors remains unclear at this time. We have previously shown with the vesicular stomatitis virus (VSV)/HCV pseudotype model that the hypervariable region 1 of the HCV E2 envelope glycoprotein helps in binding with glycosaminoglycans present on the cell surface. In this study, we have examined the binding of HCV envelope glycoproteins with chemically modified derivatives of heparin. Furthermore, we have determined the functional relevance of the interaction of heparin derivatives with HCV envelope glycoproteins for infectivity by using a human immunodeficiency virus (HIV)/HCV pseudotype, a VSV/HCV pseudotype, and cell culture-grown HCV genotype 1a. Taken together, our results suggest that the HCV envelope glycoproteins rely upon O-sulfated esters of a heparin homologue to facilitate entry into mammalian cells. 相似文献
16.
MMP-9信号肽高效诱导PEX重组蛋白在COS7细胞中分泌表达 总被引:1,自引:1,他引:1
为了便于收集和纯化, 重组蛋白常需要引导至真核细胞外。蛋白能否分泌主要取决于其是否含有信号肽, 由于不同信号肽诱导蛋白分泌的效率不同,高效信号肽的筛选已成为生物工程领域提高重组蛋白产量的重要策略之一。为了筛选诱导MMP-2 C末端PEX在COS7细胞中高效分泌表达的信号肽,在PEX的N末端分别融合大鼠生长激素(rGH)、小鼠IgG κ链和人基质金属蛋白酶-9(matrix metalloproteinase 9, MMP-9)的信号肽并比较三种信号肽引导PEX分泌表达的效率。Western免疫印迹和ELISA蛋白定量检测表明MMP-9的信号肽引导PEX蛋白分泌的效率约为其它两种信号肽的两倍。利用Ni-NTA亲和柱对细胞培养基中的PEX进行纯化,蛋白产量约为1mg/L,纯化的PEX重组蛋白具有抑制鸡尿囊膜(chorioallantoic membrane,CAM)血管发生的作用。以上结果提示MMP-9的信号肽有效诱导具有生物活性的PEX重组蛋白在COS7细胞中分泌表达。 相似文献
17.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2016,1860(8):1764-1775
BackgroundThe development of an efficient vaccine and broadly cross-neutralizing antibodies of hepatitis C virus (HCV) remains a priority. The heavily glycosylated viral envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Bacteria-derived unmethylated CpG DNA, a potent stimulator of immune cells, is important for vaccine research.MethodsHere, the immunogenicities of wild type (WT) E1E2, five N-glycosylation site mutated E1E2 glycoproteins, and five CpG-coupled E1E2 N-glycosylation mutated glycoproteins were analyzed in BALB/c mice by DNA vaccination using in vivo electroporation.ResultsThe E1E2 protein expression levels were examined and shown to be unaffected by these N-glycosylation mutations. We found that a CpG-coupled E1-N209D-E2-N430D DNA vaccine (named CpG-E1E2-M4) induced the highest cellular immune response compared to the WT E1E2, CpG-E1E2, and other mutants. Furthermore, the CpG-E1E2-M4 anti-serum effectively neutralized the infection of cell-cultured HCV (HCVcc, genotype 2a)- and HCV pseudo particles (HCVpp, genotypes 1 to 7) to Huh-7.5.1 hepatocytes. Additionally, CpG-E1E2-M4 enhanced the Interleukin-12 (IL-12) production and antigen-presenting activity of CD11c+ dendritic cells (DCs) by inducing CD4+ Th1 polarization and the production of perforin and granzyme B (GrB) in CD8+ T cells.ConclusionsAs our knowledge this is the first study revealing that the naturally poor immunogenicity of E1E2 can be enhanced by the deletion of N-glycans combined with the addition of immune activator CpG by DNA vaccination.General significanceDeletion of N-glycans can enhance viral immunogenicity. The selected CpG-E1E2-M4 mutant is a novel potential HCV DNA vaccine that elicits enhanced CD4+ Th1 and CD8+ T cell responses and neutralizing antibody production against HCV infection. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. 相似文献
18.
Evaluating replication-defective vesicular stomatitis virus as a vaccine vehicle 总被引:3,自引:0,他引:3
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We have generated replication-competent (VSV-C/E1/E2) and nonpropagating (VSVDeltaG-C/E1/E2) vesicular stomatitis virus (VSV) contiguously expressing the structural proteins of hepatitis C virus (HCV; core [C] and glycoproteins E1 and E2) and report on their immunogenicity in murine models. VSV-C/E1/E2 and VSVDeltaG-C/E1/E2 expressed high levels of HCV C, E1, and E2, which were authentically posttranslationally processed. Both VSV-expressed HCV E1-E2 glycoproteins were found to form noncovalently linked heterodimers and appeared to be correctly folded, as confirmed by coimmunoprecipitation analysis using conformationally sensitive anti-HCV-E2 monoclonal antibodies (MAbs). Intravenous or intraperitoneal immunization of BALB/c mice with VSV-C/E1/E2 or VSVDeltaG-C/E1/E2 resulted in significant and surprisingly comparable HCV core or E2 antibody responses compared to those of control mice. In addition, both virus types generated HCV C-, E1-, or E2-specific gamma interferon (IFN-gamma)-producing CD8(+) T cells, as determined by enzyme-linked immunospot (ELISPOT) analysis. Mice immunized with VSVDeltaG-C/E1/E2 were also protected against the formation of tumors expressing HCV E2 (CT26-hghE2t) and exhibited CT26-hghE2t-specific IFN-gamma-producing and E2-specific CD8(+) T-cell activity. Finally, recombinant vaccinia virus (vvHCV.S) expressing the HCV structural proteins replicated at significantly lower levels when inoculated into mice immunized with VSV-C/E1/E2 or VSVDeltaG-C/E1/E2, but not with control viruses. Our data therefore illustrate that potentially safer replication-defective VSV can be successfully engineered to express high levels of antigenically authentic HCV glycoproteins. In addition, this strategy may therefore serve in effective vaccine and immunotherapy-based approaches to the treatment of HCV-related disease. 相似文献
19.
Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates 总被引:8,自引:0,他引:8
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Heile JM Fong YL Rosa D Berger K Saletti G Campagnoli S Bensi G Capo S Coates S Crawford K Dong C Wininger M Baker G Cousens L Chien D Ng P Archangel P Grandi G Houghton M Abrignani S 《Journal of virology》2000,74(15):6885-6892
Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction. 相似文献
20.
中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)的刺突蛋白(Spike,S)亚单位1(S1)是引起宿主免疫反应和产生中和抗体的主要靶抗原,也是疫苗研发和病原检测的重要靶标,选用适宜的真核表达系统高效表达S1蛋白是进行相关研究的基础。为确定MERS-CoV S1在哺乳动物细胞中高效分泌性表达的信号肽序列,构建了含高斯荧光素酶(Gaussia luciferase,GLuc)、人组织纤溶酶原激活剂(Tissue plasminogen activator,tPA)及小鼠免疫球蛋白G的2a亚型(Mouse immunoglobular G subtype 2a,MIgG2a)7个信号肽(原始序列和改造序列)序列的MERS-CoV S1表达质粒,瞬时转染细胞后,通过Western Blot检测并比较细胞培养上清和裂解液中S1的表达水平及分泌表达效率(条带密度灰度扫描比),并对哺乳动物细胞表达的S1蛋白的纯度与抗原特性进行了分析。结果表明7种信号肽在293T、BHK21和ExpiCHO-STM三种细胞系统中介导MERS-CoV S1的高效分泌表达的效率各有不同,其中tPA-1信号肽介导S1抗原在ExpiCHO-STM中具有较高的分泌表达效率与产量,纯化的S1蛋白保持了较好的抗原性。本研究为进一步研发基于MERS-CoV S1的亚单位疫苗及免疫学检测试剂奠定了基础。 相似文献