EB病毒潜伏膜蛋白1的B细胞表位预测

目的 预测EB病毒潜伏膜蛋白1(Latent Membrane Protein 1,LMPl)的B细胞表位.方法 基于EB病毒基因组序列,采用DNAStar Lasergene软件包中的Protean软件,对LMP1的亲水性,表面可能性,抗原指数及其二级结构中的柔性区域进行分析,并结合吴玉章的抗原指数预测法预测其B细胞表位.结果 B细胞表位最有可能位于潜伏膜蛋白N端第356-358,2-19,249-314区段或其附近,而潜伏膜蛋白N端第185-223区段内或附近也可能存在B细胞表位.结论 用多参数预测EB病毒LMP1的B细胞表位,为鼻咽癌的筛查及抗肿瘤转移靶向治疗的分子免疫学研究奠定基础.     

副溶血性弧菌外膜蛋白K的B细胞线性表位预测

目的预测副溶血性弧菌外膜蛋白K(OmpK)的B细胞线性表位。方法 NCBI下载已登录的OmpK的基因序列,对其进行生物信息学分析,应用DNAStar protean软件综合分析OmpK蛋白的二级结构、柔性、表面可能性、亲水性和抗原指数等多种参数,预测其B细胞线性表位。结果 OmpK蛋白的优势B细胞线性表位位于肽链的第7-13、25-36、63-69、140-147、182-188、234-239区段。结论预测得到OmpK蛋白的6个优势B细胞线性表位,为进而克隆表达串联表位蛋白,研制副溶血性弧菌多表位疫苗奠定基础。     

SARS病毒M蛋白的二级结构和B细胞表位预测

以SARS病毒基因组序列为基础,采用GarnierRobson方法、ChouFasman方法和KarplusSchulz方法预测蛋白质的二级结构;按KyteDoolittle方案、Emini方案和JamesonWolf方案预测SARS病毒M蛋白的B细胞表位。预测结果表明,在SARS病毒M蛋白N端第11～20、27～36区段和第133～141区段可能是α螺旋中心;M蛋白分子N端第20～27、34～37,44～56,61～64,70～76,79～97,117～132,142～147,165～176区段和第216～221区段可能是β折叠中心。在M蛋白N端第5～6、40～44、105～107、112～116、189～190、202～203区段和第210～215区段具有较柔软的结构,有可能进行一定幅度的摆动或折叠而形成较复杂的三级结构。SARS病毒M蛋白N端第1～15、37～47、99～120、181～192区段和第196～215区段内或附近很可能是B细胞表位优势区域。以蛋白质的二级结构预测作为辅助手段,用抗原指数,亲水性参数和可及性参数预测SARS冠状病毒M蛋白的B细胞表位,为实验确定SARS病毒M蛋白的B细胞表位和免疫识别研究奠定了基础 。     

白念珠菌细胞壁蛋白Csp37抗原表位预测

目的预测白念珠菌细胞壁蛋白Csp37的抗原表位,分析其作为疫苗靶点的免疫原性。方法采用生物信息学方法对Csp37蛋白的抗原表位进行预测,利用ProtParam网络服务器分析蛋白基本理化性质,SignaIP 3.0预测信号肽,TMHMM软件预测跨膜区,GOR4在线分析蛋白二级结构,DNAStar预测分析亲水性、可塑性、表面可及性和氨基酸抗原指数,使用在线工具ABCPred预测B细胞抗原表位,Syfpeithi预测T细胞抗原表位。最后,综合分析B细胞和T细胞共有抗原表位。结果预测白念珠菌细胞壁蛋白Csp37的B细胞表位9个和T细胞表位8个,以及共有的优势抗原表位5个,共同优势区域为:45-48,76-78,153-158,222-225,303-305位氨基酸。结论白念珠菌细胞壁蛋白Csp37含有丰富的抗原表位,具有诱导细胞免疫应答和体液免疫应答的潜能,可以作为疫苗研究的新靶点。     

猪水疱病病毒外壳蛋白的二级结构和抗原表位预测

以SVDV外壳蛋白基因序列为基础,采用Chou-Fasman法、Garnier-Robson 法和Karplus-Schulz法预测蛋白质的二级结构;按Kyte-Doolittle方案、Emini方案和Jameson-Wolf方案预测SVDV外壳蛋白的B细胞表位。预测结果表明,SVDV外壳蛋白的二级结构较为复杂,含有较多的转角和无规则卷曲等柔性区域以及α-螺旋和β-折叠区段;SVDV外壳蛋白的VP1、VP2和VP3上均有多个区域为B细胞优势表位,其中,VP1蛋白的B细胞表位优势区域比VP2和VP3蛋白的多,与已鉴定的B细胞表位相比较,该方法预测的结果有较高的准确度。为实验确定SVDV外壳蛋白的B细胞表位和反向疫苗学设计提供理论基础。     

EB病毒包膜糖蛋白gp85真核表达载体的构建

用基因工程技术克隆EB病毒中抗原性较强的膜蛋白gp85的编码基因BXLF2,构建真核表达载体。以EB病毒B95—8细胞培养上清为模板,PCR扩增出BXLF2基因。PCR产物经SnaBⅠ和NotⅠ双酶切后克隆至毕赤酵母表达载体pPIC9K,用双酶切和DNA测序鉴定重组质粒。重组质粒双酶切的片段大小与预期符合,重组克隆外源基因的测序结果与献报道一致。结果表明,EB病毒gp85的编码基因BXLF2被成功地克隆入真核表达载体pPIC9K,为下一步在毕赤酵母中表达EB病毒gp85蛋白建立了基础。     

A型肉毒神经毒素(BoNT/A)基因序列分析及其B细胞表位预测

比较GenBank中4个不同毒株的A型肉毒神经毒素（BoNT／A）的基因组序列,发现其基因序列的一致性高迭92．2％-99．9％。基于保守性高的BoNT／A氨基酸序列,根据BioSun和LaserGene软件包中的表住分析相关参数,辅以对BoNT／A蛋白的二级结构的分析,综合预测BoNT／A的B细胞表位。结果表明,BoNT／A轻链的142-150、284-292区段,轻链的284-292,重链的440-450、465-480、538-549、699-710、751-760、1087-1095、1224-1231、1263-1270区段是B细胞表位优势区的可能性较大。多参数方案井结合不同软件综合预测BoNT／A蛋白的B细胞表位,为进一步实验鉴定BoNT／A的B细胞表位及其多表位疫苗设计和研究奠定了基础。     

金黄色葡萄球菌肠毒素A蛋白的B细胞表位的预测

目的预测金黄色葡萄球菌肠毒素A蛋白（SEA）的B细胞表位。方法以金黄色葡萄球菌合肥乳源分离株M3基因组DNA为模板,PCR扩增SEA基因并进行序列测定与分析。应用DNAstar protean软件对SEA蛋白的二级结构、柔性、亲水性、表面可能性和抗原指数等多参数进行综合分析,预测其B细胞表位。结果M3分离株的SEA基因全长774bp,编码由257个氨基酸组成的相对分子量为29．67kDa的SEA蛋白,M3分离株SEA基因与标准株的核苷酸序列与氨基酸序列同源性分别为98．7％和98．4％。SEA蛋白的优势B细胞表位位于肽链的第64—68、100～107、138—141、156—160、166～173、213～217和237～244区段。结论预测出SEA蛋白的7个优势B细胞表位,为进而克隆表达表位蛋白,制备针对SEA表位的单克隆抗体奠定了基础。     

猪肌生成抑制素基因去信号肽蛋白二级构预测和B细胞表位分析

目的预测猪肌生成抑制素去信号肽蛋白的二级结构和B细胞优势抗原表位,为生产该蛋白的单克隆抗体、建立噬菌体抗体库、研制针对该基因的表位多肽疫苗、表位核酸疫苗等奠定基础。方法根据猪肌生成抑制素去信号肽蛋白氨基酸序列,应用7种参数和方法分析预测二级结构和抗原表位,包括Garnier-Robson、Chou-Fasman、Karplus-Schulz、Kyte-Doolittle、Emini、Jameson-Wolf及吴氏综合预测方法。结果MSTN去信号肽蛋白存在多个潜在的抗原表位位点,其中B细胞抗原优势表位可能在1-11、41-55、57-64、62-90、99-104、138-144、193-200、202-212、235-243区段或其附近,此结果将为进一步鉴定和合成多肽疫苗和表位核酸疫苗制备抗猪MSTN蛋白抗体提供依据,并为研究MSTN结构和功能奠定基础。     

山羊痘病毒P32基因序列分析及其B细胞表位预测

目的：比较山羊痘病毒不同分离株间P32基因的同源性,并对其B细胞表位进行预测。方法：选取GenBank上10株不同山羊痘毒株P32基因序列,采用DNAsis MAX序列分析软件对该基因序列进行同源性分析,并以B细胞表位分析参数及蛋白质二级结构分析数据,综合预测P32蛋白B细胞表位。结果：10株不同山羊痘毒株P32基因核苷酸及氨基酸序列同源性分别为99.4%和98.4%;在P32蛋白的肽链中,19-60、64-80、98-118、138-182和214-275区段亲水性强,17-45、64-75、88-97、110-128、152-165和201-251区段柔韧性好,150-172、200-255区段抗原指数高,而31-59、101-119、142-145、165-172和215-252区段表面可及性高,42-56、159-181、200-208和227-259区段。结论：不同山羊痘病毒株间P32基因具有较高的同源性,P32蛋白227-251区段可能是B细胞表位优势区,为P32蛋白功能的深入研究与新型疫苗的设计提供一定的基础。     

Inhibition of Epstein-Barr virus (EBV) release from P3HR-1 and B95-8 cell lines by monoclonal antibodies to EBV membrane antigen gp350/220.

Antibody-mediated inhibition of Epstein-Barr virus (EBV) release from the EBV-productive cell lines P3HR-1 and B95-8 was probed with two monoclonal antibodies (MAbs), 72A1 and 2L10, which immunoprecipitated the same EBV membrane antigen (MA) gp350/220 found with the 1B6 MAb with which inhibition of EBV release from P3HR-1 cells was first described. These three MAbs were not equivalent in either MA reactivities or functional effects, reflecting the variable expression of different epitopes of gp350/220. 1B6 recognized MA on P3HR-1 cells, which expressed predominately the gp220 form of MA. 1B6 did not recognize (or barely recognized) a determinant on B95-8 cells. MAbs 2L10 and 72A1 reacted as well with B95-8 cells as they did with P3HR-1 cells. MAbs 1B6 and 2L10 neutralized neither P3HR-1 nor B95-8 virus, but 72A1 neutralized both viruses. MAbs 1B6 and 72A1 inhibited P3HR-1 virus release, as measured by the assay for infectious virus and by DNA hybridization analysis of released virus, but 2L10 had no such activity. 72A1 (but not 1B6) inhibited release of EBV from B95-8 cells. These experiments pointed to the presence of three different epitopes on gp350/220, identified with the respective MAbs and having varying involvement in virus neutralization and virus release inhibition.     

Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo.

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.     

EBV structural antigens, gp350 and gp85, as targets for ex vivo virus-specific CTL during acute infectious mononucleosis: potential use of gp350/gp85 CTL epitopes for vaccine design

For many years, EBV vaccine development efforts have concentrated on the use of structural Ag, gp350, and have been directed toward Ab-mediated blocking virus attachment to the target cell. There is increasing evidence to suggest that the development of neutralizing Abs in vaccinated animals does not always correlate with protection; nevertheless, it has been postulated that gp350-specific T cell-mediated immune responses may have an effector role in protection. This hypothesis has largely remained untested. In the present study, we demonstrate that CTL from acute infectious mononucleosis patients display strong ex vivo reactivity against the EBV structural Ags, gp85 and gp350. Moreover, long-term follow up studies on infectious mononucleosis-recovered individuals showed that these individuals maintain gp350- and gp85-specific memory CTL, albeit at low levels, in the peripheral blood. These results strongly suggest that CTL specific for EBV structural proteins may play an important role in the control of EBV infection during acute infection. More importantly, we also show that prior immunization of HLA A2/Kb transgenic mice with gp350 and gp85 CTL epitopes induced a strong epitope-specific CTL response and afforded protection against gp85- or gp350-expressing vaccinia virus challenge. These results have important implications for future EBV vaccine design and provides evidence, for the first time, that CTL epitopes from EBV structural proteins may be used for establishing strong antiviral immunity against EBV infection.     

Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2)

The Epstein-Barr virus gp350/220 envelope protein mediates virus attachment to the EBV/C3dg receptor (CR2) of human B lymphocytes. Synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement C3dg protein, were used to identify a receptor binding epitope. A peptide corresponding to the N terminus of gp350/220, EDPGFFNVE, bound to purified CR2 and to CR2 positive but not CR2 negative B and T lymphoblastoid cell lines. Soluble monomeric gp350/220 peptide blocked CR2 binding to immobilized EBV, while multimeric forms of the N-terminal gp350/220 peptide conjugated to albumin efficiently blocked recombinant gp350/220 and C3dg binding to B cells as well as EBV-induced B cell proliferation and transformation. These studies indicate that the N-terminal region of gp350/220 plays a crucial role in mediating the earliest stages of EBV infection of B cells and provides a molecular basis for the restricted host cell EBV tropism.     

Diverse IgG serum response to novel glycopeptide epitopes detected within immunodominant stretches of Epstein-Barr virus glycoprotein 350/220: diagnostic potential of O-glycopeptide microarrays

The Epstein-Barr virus (EBV) envelope glycoprotein 350/220 (gp350/220) is the most abundant molecule on the viral surface and it is responsible for the initial viral attachment to cell surface of the host. As many other viral envelope proteins, it is highly glycosylated, not least with O-linked glycans, most of which essential for EBV life cycle. EBV gp350/220 is also a primary target for neutralizing antibodies in the human hosts and a promising candidate for an EBV vaccine. Here we showed that recombinant GalNAc transferases can glycosylate scan peptides of the EBV gp350/220 envelope protein immobilized on microarray glass slides. We also identified serum IgG antibodies to a selection of peptides and O-glycopeptides, whereas sera from EBV-IgG negative individuals remained negative. We here describe novel glycopeptide epitopes present within immunodominant stretches of EBV gp350/220 and demonstrate a remarkable variability between individual samples with respect to their reactivity patterns to peptides and glycopeptides. The study provides additional insights into the complex B-cell response towards the EBV gp350/220 envelope protein, which may have implications for diagnostic and vaccine developments.     

Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells.

Entry of an enveloped virus such as Epstein-Barr virus (EBV) into host cells involves fusion of the virion envelope with host cell membranes either at the surface of the cell or within endocytic vesicles. Previous work has indirectly implicated the EBV glycoprotein gp85 in this fusion process. A neutralizing monoclonal antibody to gp85, F-2-1, failed to inhibit binding of EBV to its receptor but interfered with virus fusion as measured with the self-quenching fluorophore octadecyl rhodamine B chloride (R18) (N. Miller and L. M. Hutt-Fletcher, J. Virol. 62:2366-2372, 1988). To test further the hypothesis that gp85 functions as a fusion protein, EBV virion proteins including or depleted of gp85 were incorporated into lipid vesicles to form virosomes. Virosomes were labeled with R18, and those that were made with undepleted protein were shown to behave in a manner similar to that of R18-labeled virus. They bound to receptor-positive but not to receptor-negative cells and fused with Raji cells but not with receptor-positive, fusion-incompetent Molt 4 cells; monoclonal antibodies that inhibited binding or fusion of virus inhibited binding and fusion of virosomes, and virus competed with virosomes for attachment to cells. In contrast, virosomes made from virus proteins depleted of gp85 by immunoaffinity chromatography remained capable of binding to receptor-positive cells but failed to fuse. These results are compatible with the hypothesis that gp85 is actively involved in the fusion of EBV with lymphoblatoid cell lines and suggest that the ability of antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on the function of gp85 as a fusion protein.     

Epstein-Barr virus membrane antigens: characterization, distribution, and strain differences.

The Epstein-Barr virus (EBV)-associated membrane antigen polypeptides (350,000, 220,000, 140,000, and 85,000 daltons) are recognized by a rabbit anti-EBV serum and are present on the plasma membranes of producer cell lines, as we demonstrated previously. In this report, we show that these polypeptides are present on intact virus particles. Subcellular fractionation revealed that these antigens are distributed throughout the cell, except for the 85,000-dalton protein, which was poorly represented in the nuclear fraction. In addition, an EBV-associated protein of 160,000 daltons, which comigrates with a major component of the viral capsid, was detected in the cytoplasmic and nuclear fractions. The immunoprecipitation patterns of 13 different EBV isolates were similar, with two exceptions. First, the 350,000- and 220,000-dalton polypeptides from marmoset cell lines had slightly larger molecular sizes than the corresponding polypeptides from human cell lines. Second, B95-8 virus and B95-8-derived human and marmoset cell lines contained little of the 220,000-dalton protein; however, 883L, the human parent line of B95-8, has a normal amount of the 220,000-dalton protein. Thus, the B95-8 strain of EBV appears to be a structurally defective variant. We have not observed any variation in protein patterns associated with different EBV disease states. The 350,000-, 220,000-, and 85,000-dalton polypeptides were shown to be glycoproteins by incorporation of [3H]mannose and [3H]glucosamine and to contain N-asparagine-linked glycosyl groups by their sensitivity to tunicamycin. To simplify future work, the following nomenclature for these EBV-associated polypeptides is suggested: 350,000 (gp350), 220,000 (gp220), 160,000 (p160), 140,000 (p140), and 85,000 (gp85).     

Mapping of B-cell epitopes on the polypeptide chain of the Epstein-Barr virus major envelope glycoprotein and candidate vaccine molecule gp340.

The Epstein-Barr virus (EBV) major envelope glycoprotein gp340 is the subject of current efforts to develop an EBV subunit vaccine. The importance of gp340-specific humoral immunity has been highlighted by studies of natural infection in humans and gp340 immunization of experimental animals. The former studies have demonstrated the presence of gp340-specific serum antibodies which mediate EBV neutralization, complement fixation, and antibody-dependent cellular cytotoxicity. The latter studies have often shown a correlation between the induction of gp340-specific EBV-neutralizing antibodies and protection from virus challenge. We have used a series of bacterial beta-galactosidase-gp340 fusion proteins and overlapping synthetic peptides from the gp340 open reading frame to map the positions of B-cell epitopes within the gp340 primary amino acid sequence. The data reported here indicate the presence of B-cell epitopes within the carboxy-terminal third of the gp340 polypeptide chain. These epitopes could not be detected with a peptide enzyme-linked immunosorbent assay, thereby suggesting that they are discontinuous. Affinity purification of antibodies with a gp340 fusion protein from the carboxy terminus of the gp340 polypeptide chain has been used to show that these antibodies are not EBV neutralizing in vitro. The consequences of these findings for future EBV vaccine development are considered.     

Activation of the alternative complement pathway by EBV and the viral envelope glycoprotein, gp350

The EBV-producing B lymphoblastoid cell line B95-8 was found to efficiently activate the alternative C pathway whether assessed with Mg-EGTA-treated human serum or with mixtures of the purified proteins of the pathway (PAP). The ability of the cells to activate was markedly increased after stimulation of EBV replication by treatment of the cells with a phorbol ester, and decreased by treatment of the cells with a viral polymerase inhibitor. Alternative pathway activation was dependent on the presence of either properdin or EBV-immune IgG; the addition of either alone to the PAP led to the deposition of 200,000 C3 molecules/cell. The addition of both properdin and immune IgG to the PAP markedly increased C3 binding to a level of 800,000 molecules/cell. Several lines of evidence indicate that the major external glycoprotein of EBV, gp350, mediates alternative pathway activation by B95-8 cells. First, the ability to activate C positively correlated with gp350 expression on the surface of the EBV-producing cells and gp350- cells failed to activate; second, the anti-EBV antibody in immune human sera which enhanced activation specifically immunoprecipitated gp350 from membranes of B95-8 cells; third, a significant proportion of the C3 which became bound to the cells during activation was attached either to gp350 or to the anti-gp350 antibody found in immune human sera; and fourth, purified gp350, as well as EBV, efficiently activated the alternative pathway. These results indicate that gp350, an EBV envelope glycoprotein, is an efficient alternative pathway activator and its expression on cell membranes is associated with the ability to activate C.