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.     

EB病毒gp125蛋白的B细胞表位预测

目的预测EB病毒gp125蛋白的B细胞表位。方法基于EB病毒gp125蛋白的氨基酸序列,采用亲水性参数、可及性参数、极性参数和抗原性指数方案等,辅以对gp125蛋白的二级结构中的柔性区域的分析,预测gp125蛋白的B细胞表位。结果最有可能的B细胞表位位于gp125蛋白N端第403-416、565—574、578—584、618-630和832—843区段及其附近。结论用多参数预测EB病毒gp125蛋白的B细胞表位,为制备具有高灵敏度和高特异性的鼻咽癌诊断试剂及研究抗肿瘤转移靶向治疗的分子免疫学奠定基础。     

Analysis of antibody response to synthetic peptides derived from the fusion protein of measles virus

A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.     

The antigenicity of tobacco mosaic virus

The antigenic properties of the tobacco mosaic virus (TMV) have been studied extensively for more than 50 years. Distinct antigenic determinants called neotopes and cryptotopes have been identified at the surface of intact virions and dissociated coat protein subunits, respectively, indicating that the quaternary structure of the virus influences the antigenic properties. A correlation has been found to exist between the location of seven to ten residue-long continuous epitopes in the TMV coat protein and the degree of segmental mobility along the polypeptide chain. Immunoelectron microscopy, using antibodies specific for the bottom surface of the protein subunit, showed that these antibodies reacted with both ends of the stacked-disk aggregates of viral protein. This finding indicates that the stacked disks are bipolar and cannot be converted directly into helical viral rods as has been previously assumed. TMV epitopes have been mapped at the surface of coat protein subunits using biosensor technology. The ability of certain monoclonal antibodies to block the cotranslational disassembly of virions during the infection process was found to be linked to the precise location of their complementary epitopes and not to their binding affinity. Such blocking antibodies, which act by sterically preventing the interaction between virions and ribosomes may, when expressed in plants, be useful for controlling virus infection.     

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。     

Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.     

Generation of antiserum to specific epitopes

The ability to prevent disease by immunization with subunit vaccines that incorporate specific epitopes was demonstrated by DiMarchi et al. (1), who used a synthetic peptide to protect cattle against foot-and-mouth disease. However, generation of antibody to peptide antigens is often difficult owing to the small molecular mass and limited chemical complexity. We tested the hypothesis that recombinant DNA and synthetic peptide techniques would make it possible to stimulate vigorous immune responses to specific epitopes of an outer membrane protein ofNeisseria gonorrhoeae. The MtrC AP1 sequence from the invariant mtrC gonococcal lipoprotein was genetically fused to maltose binding protein. The resultant fusion protein was used as the primary immunogen to stimulate MtrC AP1-specific antiserum. To enhance antibody production specific to MtrC AP1, boosting immunizations were performed with synthetic MtrC AP1 sequence contained in a multiple antigenic peptide system immunogen. The MtrC AP1-specific antiserum strongly recognized the MtrC protein on Western blots and appeared to bind native MtrC proteinin situ. The generation of antibody in this fashion provides the technology to produce antibody to defined epitopes of any protein, including those found in the gonococcal outer membrane. The ability of those antibodies to inhibit bacterial growth or to activate complement protein can then be tested.     

沙眼衣原体多形态膜蛋白D基因序列分析及其B细胞抗原表位预测

分析沙眼衣原体多形态膜蛋白D(PmpD)的基因序列并预测PmpD蛋白的B细胞抗原表位。在GenBank中检索沙眼衣原体不同血清型的PmpD基因序列,进行序列比对分析。以L2血清型PmpD基因序列为材料,采用Karplus-Schulz、Chou-Fasman和Gamier-Robson方案预测蛋白质的二级结构和柔性区;按Jamesonv-Wolf方案预测抗原指数,运用Kyte-Doolittle方案预测PmpD氨基酸的亲水性,利用Emini方案预测蛋白质的表面可及性。检索到20个沙眼衣原体不同菌株的PmpD基因序列,分析发现其核苷酸序列非常保守,一致性高达99.14%~100%;对预测结果综合分析,推测最有可能的B细胞表位位于PmpD N端的67~74、132~140、335~340、851~861、972~988及1091~1097。多参数方案综合预测PmpD蛋白的B细胞抗原表位,为进一步实验鉴定PmpD抗原表位及其多表位疫苗设计和研究奠定基础。     

Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions.

Independent isolates of human immunodeficiency virus (HIV) exhibit a striking genomic diversity, most of which is located in the viral envelope gene. Since this property of the HIV group of viruses may play an important role in the pathobiology of the virus, we analyzed the predicted amino acid sequences of the envelope proteins of seven different HIV strains, three of which represent sequential isolates from a single patient. By using a computer program that predicts the secondary protein structure and superimposes values for hydrophilicity, surface probability, and flexibility, we identified several potential antigenic epitopes in the envelope proteins of the seven different viruses. Interestingly, the majority of the predicted epitopes in the exterior envelope protein (gp120) were found in regions of high sequence variability which are interspersed with highly conserved regions among the independent viral isolates. A comparison of the sequential viral isolates revealed that changes concerning the secondary structure of the protein occurred only in regions which were predicted to be antigenic, predominantly in highly variable regions. The membrane-associated protein gp41 contains no highly variable regions; about 80% of the amino acids were found to be conserved, and only one hydrophilic area was identified as likely to be accessible to antibody recognition. These findings give insight into the secondary and possible tertiary structure of variant HIV envelope proteins and should facilitate experimental approaches directed toward the identification and fine mapping of HIV envelope proteins.     

Getting in Touch with the Clathrin Terminal Domain

The N‐terminal domain (TD) of the clathrin heavy chain is folded into a seven‐bladed β‐propeller that projects inward from the polyhedral outer clathrin coat. As the most membrane‐proximal portion of assembled clathrin, the TD is a major protein–protein interaction node. Contact with the TD β‐propeller occurs through short peptide sequences typically located within intrinsically disordered segments of coat components that usually are elements of the membrane‐apposed, inner ‘adaptor’ coat layer. A huge variation in TD‐binding motifs is known and now four spatially discrete interaction surfaces upon the β‐propeller have been delineated. An important operational feature of the TD interaction sites in vivo is functional redundancy. The recent discovery that ‘pitstop’ chemical inhibitors apparently occupy only one of the four TD interaction surfaces, but potently block clathrin‐mediated endocytosis, warrants careful consideration of the underlying molecular basis for this inhibition.     

ZNF185基因抗原表位分析及合成肽免疫避孕效果研究

目的:预测ZNF185基因的抗原表位,并研究相应合成多肽诱导的免疫应答和免疫避孕效应。方法:应用DNA Star软件预测ZNF185蛋白二级结构、亲水性、可塑性、表面可及性与抗原指数。合成携带ZNF185抗原表位的多肽,进行小鼠免疫实验,检测小鼠血清中IgG抗体水平、交配率、妊娠率及每窝产仔数,并观察生殖器官是否发生病变。结果:筛选ZNF185两段氨基酸序列合成多肽,该多肽能够诱导小鼠产生较高的抗体水平,实验组小鼠交配率和妊娠率显著低于对照组,生殖器官并无发生明显的组织病理学变化。结论:ZNF185具有一定的抗生育作用。     

An immunodominant site of γ-zein1 is in the region of tandem hexapeptide repeats

The immunochemical data from studies with polyclonal antisera to γ-zein₁, the 27 kD component of the maize prolamin, indicated that the region containing 8 tandem repeats of the sequence PPPVHL is an immunodominant site. In one case, the entire antibody repertoire of an antiserum recognized epitope(s) within this region. Three 17-mer oligopeptides corresponding to the predicted antigenic epitopes of γ-zein₁ were synthesized and reacted with three different anti-γ-zein₁ sera in order to map antigenic sites in the intact protein. These antisera yielded positive reactions with a 17-mer peptide (peptide 37), which was not in a hydrophilic maximum but derived from the repeat region. The same antisera gave little or no reaction with other peptides (peptides 38 and 39), both of which were in a hydrophilic maximum. In addition, an antiserum to peptide 37 reacted strongly with both the homologous antigen and the intact γ-zein₁. Peptide 37 also blocked the binding of antisera to γ-zein₁ in competition assays. Subsequently, the shorter 6-mer (peptide 82) and 12-mer (peptide 80) versions of peptide 37 were synthesized, and both reacted with anti-peptide 37 serum and also with each of the three anti-γ-zein₁ sera. In these reactions and in competition assays, the reactivity and the blocking ability increased in proportion to the length of the peptide. Based on these data, it was concluded that the repeat region of γ-zein₁ is the site of one or more continuous immunodominant epitopes. The data also suggest that the repeat region is exposed on the surface of the folded protein and probably occur as a mobile, random coil.     

A major part of the polypeptide chain of tobacco mosaic virus protein is antigenic

Eighteen synthetic peptides representing virtually the entire length of the polypeptide chain of tobacco mosaic virus coat protein (TMVP) have been analyzed for their ability to bind in an enzyme immunoassay to 30 monoclonal antibodies raised against the dissociated viral subunits. Only five of the monoclonal antibodies were able to bind a number of peptides while the other 25 antibodies recognized only the complete molecule and seemed to be specific for conformational features that are absent in the peptide fragments. The 18 peptides were also tested for their ability to bind to several antisera to TMVP. Virtually the entire sequence of TMVP possessed antigenic activity. Four new epitopes were identified in the vicinity of residues 19–32, 90–95, 115–134 and 134–146. These results bring to 11 the number of continuous epitopes that have been identified in the TMVP molecule and show that the entire surface of the molecule is antigenic. When peptides of TMVP of a length of 6–8 residues were tested for antigenic activity previously a correlation was found between the location of short continuous epitopes and mobile segments of the protein. In the present study, in which longer peptides as well as monoclonal antibodies were used to probe the antigenicity of TMVP, additional conformation-dependent epitopes were shown to be present. Our results illustrate the operational nature of any definition of antigenicity and caution against the use of any single criterion for distinguishing between antigenic and non-antigenic regions of a protein.     

Cytopathology of Apple stem pitting virus in Nicotiana occidentalis L.

Apple stem pitting virus (ASPV) is a causal agent of stem pitting associated disease in pomes fruit trees. The present report focuses on a cytopathological effect of ASPV infection in a herbaceous host Nicotiana occidentalis ‘37B’. A leaf dip preparation shows predominantly basic virus particles and aggregated particles 800 nm and 3200 nm long respectively. The main cytopathological effect observed in ASPV infected N. occidentalis includes fibrous aggregates of virus particles (massive/or few), formation of membranous vesicles and proliferation of the endoplasmic reticulum.     

First Report of Dragon Fruit (Hylocereus undatus) Anthracnose Caused by Colletotrichum truncatum in China

Anthracnose disease was detected from dragon fruit (Hylocereus undatus) at a market of Yuanjiang County, Yunnan Province, China. The results of pathogenicity test, morphology studies and sequence analyses based on ITS and β‐tubulin loci indicated that the disease was caused by Colletotrichum truncatum. The pathogen produced elliptic, yellow spots with chlorotic halos on the surface of the fruit, and the lesion become depressed gradually. Grey to black acervuli appeared on the lesion surface in concentric circles later. This is the first report of dragon fruit anthracnose caused by this pathogen in China.     

Occurrence and Diversity of Pome Fruit Viruses in Apple and Pear Orchards in Latvia

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus are economically important viruses infecting fruit tree species worldwide. To evaluate the occurrence of these pome fruit viruses in Latvia, a large‐scale survey was carried out in 2007. Collected samples were tested for infection by DAS ELISA and multiplex RT‐PCR. The accuracy of the detection of the viruses in multiplex RT‐PCR was confirmed by sequencing amplified PCR fragments. The results showed a wide occurrence of viruses in apple and pear commercial orchards established from non‐tested planting material. More than 89% of the tested apple trees and more than 60% of pear trees were infected with one or more pome fruit viruses. Analyses showed that the high occurrence of viruses in several apple cultivars is due to the propagation of infected clonal rootstocks and scions from infected mother trees. Sequence analyses targeting the 3′‐terminal region of the tested viruses showed various degrees of genetic diversity within respective virus isolates. This is the first report of the occurrence of ACLSV, ASGV and ASPV in apple and pear trees in Latvia and demonstrates their genetic diversity in different host genotypes.     

轮状病毒LLR株VP7抗原表位的预测与验证

在GenBank中检索A组轮状病毒不同血清型的VP7基因信息,在氨基酸水平上与G10血清型LLR株的VP7序列进行序列对比,分析其血清型特异的氨基酸保守序列位点。结合蛋白质二级结构预测理论方案,设计合成3条具有轮状病毒G10血清型特异性氨基酸序列的多肽。通过检测合成肽对轮状病毒免疫血清与LLR抗原的结合抑制,证实三条多肽均具备了LLR表位属性。     

BuildBeta—A system for automatically constructing beta sheets

We describe a method that can thoroughly sample a protein conformational space given the protein primary sequence of amino acids and secondary structure predictions. Specifically, we target proteins with β‐sheets because they are particularly challenging for ab initio protein structure prediction because of the complexity of sampling long‐range strand pairings. Using some basic packing principles, inverse kinematics (IK), and β‐pairing scores, this method creates all possible β‐sheet arrangements including those that have the correct packing of β‐strands. It uses the IK algorithms of ProteinShop to move α‐helices and β‐strands as rigid bodies by rotating the dihedral angles in the coil regions. Our results show that our approach produces structures that are within 4–6 Å RMSD of the native one regardless of the protein size and β‐sheet topology although this number may increase if the protein has long loops or complex α‐helical regions. Proteins 2010. © Published 2009 Wiley‐Liss, Inc.     

Detection of expansin proteins and activity during tomato fruit ontogeny

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.     

An immunological study of corrinoid proteins from bacteria revealed homologous antigenic determinants of a soluble corrinoid-dependent methyltransferase and corrinoid-containing membrane proteins from Methanobacterium species

The hypothesis of common epitopes in corrinoid-dependent enzymes was tested by a monospecific polyclonal antiserum against the 33 kDa corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum Marburg. Cross-reaction was detected with the 33 kDa and the 31 kDa subunits of the corrinoid-containing enriched 5-methyl-H₄MPT: 5-hydroxybenzimidazolyl cobamide methyltransferase from the cytoplasmic fraction and a 33 kDa protein from the membrane fraction of Methanobacterium thermoauto-trophicum H. This indicates that both proteins have similar antigenic determinants and that they may have similar function as methyltransfer proteins. Also a soluble 20 kDa protein of yet unknown function from Clostridium barkeri cross-reacted with the antiserum. No cross-reactions were observed with the purified corrinoid-containing 2-methyleneglutarate mutase from C. barkeri, the corrinoid/iron-sulfur protein from C. thermoaceticum, the carbon monoxide dehydrogenases from C. thermoaceticum and Methanothrix soehngenii, and the corrinoid-binding protein intrinsic factor from porcine gastric mucosa. Also cell extracts from the corrinoid-rich bacteria Sporomusa ovata, Methanolobus tindarius, Chloroflexus aurantiacus, Propionibacterium shermanii, the membrane fraction and the cytoplasmic fraction of Methanococcus voltae or extracts from human liver, contained no antibody combining sites others than with the preimmunological serum. These findings indicate, that many corrinoid-containing proteins from bacteria have no common antigenic determinants.Abbreviations CH₃-H₄MPT N ⁵-methyl-tetrahydromethanopterin - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - ELISA enzyme linked immunosorbent assay - DSM Deutsche Sammlung von Mikroorganismen