An immunoglobulin y that specifically binds to an in silico-predicted unique epitope of Zika virus non-structural 1 antigen

Objective: To identify unique immunogenic epitopes of Zika virus non-structural 1 (NS1) antigen and produce immunoglobulin Y (IgY) for potential use in he diagnosis of of Zika virus infection. Methods: Immunogenic epitopes were identified using in silico B-cell epitope prediction. A synthetic peptide analog of the predicted epitope was used to induce antipeptide IgY production in hens which was purified using affinity chromatography. Presence of purified IgY and its binding specificity were performed by gel electrophoresis and ELISA, respectively. Results: Out of the nine continuous epitopes identified, the sequence at position 193-208 (LKVREDYSLECDPAVI) was selected and used to produce anti-peptide IgY. The produced IgY was found to bind to the synthetic analog of the Zika virus NS1 immunogenic epitope but not to other flaviviruses and random peptides from other pathogens. Conclusions: In this study, we identified an immunogenic epitope unique to Zika virus that can be used to develop a serodiagnostic tool that specifically detect Zika virus infection.

Analysis of lactate dehydrogenase gene polymorphisms and prediction of B cell epitopes in four human Plasmodium species / 中国血吸虫病防治杂志

Objective To analyze the polymorphism of Plasmodium lactate dehydrogenase (pLDH) gene and predict B-cell epitopes in pLDH peptides in four species of human malaria parasites. Methods The blood samples and epidemiological characteristics were collected from malaria cases in Yunnan Province registered in the National Notifiable Disease Report System. The pLDH genes of four human Plasmodium species were amplified using nested PCR assay and sequenced. The polymorphisms of pLDH genes was analyzed using the software MEGA version 7.0.26 and DnaSP version 5.10, and the B-cell epitopes were predicted in pLDH peptides using the Immune Epitope Database (IEDB). Results The sequences of P. vivax LDH (PvLDH), P. falciparum LDH (PfLDH), P. ovale LDH (PoLDH) and P. malariae LDH (PmLDH) genes were obtained from 153, 29, 17 and 11 blood samples from patients with P. vivax, P. falciparum, P. ovale and P. malariae malaria, respectively, which included 15, 2, 4 and 2 haplotypes and had a nucleotide diversity (π) of 0.104. A high level of intra-species differentiation was seen in the PoLDH gene (π = 0.012), and the π values were all < 0.001 for PvLDH, PfLDH and PmLDH genes. Active regions of B-cell antigen were predicted in the pLDH peptide chain of four human malaria parasites, of 4 to 5 in each chain, and the activity score was approximately 0.430. Among these peptide chains, the “86-PGKSDKEWNRD-96” short-peptide was a B-cell epitope shared by all four species of human malaria parasites, and the “266-GQYGHS (T)-271” short-peptide was present in PvLDH and PoLDH peptide chains, while “212-EEVEGIFDR-220” was only found in the PvLDH peptide chain, and “208-LISDAE-213” was only seen in the PfLDH peptide chain. Conclusions The PoLDH gene polymorphism may be derived from the weak negative purification selection, while PvLDH, PfLDH and PmLDH genes may maintain a relatively conservative state. There may be two B-cell epitopes “212-EEVEGIFDR-220” and “208-LISDAE-213” in the proximal region of the C terminal in the pLDH peptide chain, which is feasible to differentiate between P. vivax and P. falciparum infections.

Prokaryotic expression of the GapC protein of Streptococcus uberis and prediction, identification of its B-cell epitopes / 生物工程学报

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide 266AANDSYGYTEDPIVSSD282 reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.

Polymorphism of P66 in / 生物医学与环境科学（英文）

Objective@#To study the polymorphism in P66 and its human B-cell epitopes of @*Methods@#Polymerase chain reaction (PCR) and sequencing were used to obtain the P66 sequences of 59 Chinese @*Results@#Results showed that genetic and amino acid diversity presented in the 66 kD protein of all 59 Chinese strains, especially in @*Conclusion@#In P66 of 59 Chinese strains, polymorphisms were widely distributed. More importantly, the P66 amino acid sequences of

Study of B cell epitope conserved region of the Zika virus envelope glycoprotein to develop multi-strain vaccine

A Zika virus (ZIKV) infection causes severe clinical manifestations, both in newborn baby and adult. It was consideredas the public health emergency by the World Health Organization in 2016. Until now, there is no approved drug orvaccine for the treatment or prevention of multi-strain ZIKV infection. The present work attempts to identify theB cell epitope conserved region of ZIKV envelope glycoprotein through an intensive in silico study. A total of 363ZIKV strains was retrieved from the National Center of Biotechnology Information (NCBI) database, then aligned toobtain the conserved region of ZIKV glycoprotein. The interaction between the conserved region with Axl receptortyrosine kinase, a ZIKV receptor on the host cell, has been evaluated through molecular docking approach. TheB cell epitope was identified using the immune epitope database (IEDB) web server. This study revealed that theconserved region of ZIKV envelope glycoprotein interacts with Asp9, Glu12, Glu40, Asp177, Glu243, and Glu245of Axl receptor. Two sequences in ZIKV envelope glycoprotein, “ISDSDSRCPTQGEALKQSDTQY” (22-mer) and“SQHSGMGETDERAKVETPNSPRAEATL” (27-mer), are identified as B cell epitopes. Further studies are necessaryto confirm their possibility as the potential ZIKV multi-strain vaccine in the future.

Homology modeling and epitope prediction of Der f 33

Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34-45, 63-67, 103-108, 224-230, 308-316, and 365-377) and four T cell epitopes (positions 178-186, 241-249, 335-343, and 402-410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.

Prediction and identification of immunodominant linear B cell epitopes in N9 of avian influenza virus H7N9 / 中华实验和临床病毒学杂志

Objective@#To identify immunodominant linear B cell epitopes in neuraminidase of avian influenza virus H7N9.@*Methods@#By using protean algorithms of bioinformatic software DNAStar, antigenicity, hydrophilicity and surface probability of the H7N9 neuraminidase sequence was analyzed and their corresponding average indexes were calculated. Multiple regions containing potential linear B cell epitopes were predicted. Corresponding peptides were synthesized artificially and used in peptide-ELISA individually to check their reactivity to confirmed H7N9 positive human sera, and H7N9 negative human sera was used as control.@*Results@#Seven potential linear B cell epitopes, namely A to G, were predicted according to their relatively strong antigenicity, hydrophilicity and surface probability. All corresponding synthetic peptides reacted strongly with H7N9 positive sera.@*Conclusions@#Immunodominant linear B cell epitopes in neuraminidase of H7N9 were successfully predicted and confirmed. It will facilitate to clarify molecular basis of the antigen specificity and to make respective antibodies.

Structures and Antigenic Epitopes of Dust Mite Allergens: Der p 2 and Der f 2: a Comparative Study / 现代医院

Objective To compare and analyze the primary and secondary structures and antigenic epitopes of the two allergens: Der p 2 and Der f 2. Methods The protein sequences of Der p 2 and Der f 2 were downloaded online. The primary and secondary structures of the dust mite allergens were compared and analyzed bioinformatically to determine the potential epitope and signal peptide sites. Results Both Der p 2 and Der f 2 contained 146 amino acids and 9 potential protein binding sites with a secondary structure that mainly contains [3 - sheets, and there might be signal peptides site at the 1st 17th segment of the N - terminus. B cell epitopes analysis revealed that both Der p 2 and Der f 2 have 9 potential linear B epitopes and 2 conformational B epitopes. NetMHCⅡserver prediction showed Der p 2 contains 6 high affinity sites, whereas Der f 2 0nly contains 5. Conclusion This study may lay the foundation for further research of the biochemical function of the 2 allergens and contribute to vaccine development for allergen - specific immunotherapy.

Bioinformatics analysis of VP1 protein of coxsackievirus A6 / 中国免疫学杂志

Objective:To predict the basic physicochemical properties ,structure and function ,and linear B-cell epitopes of the capsid protein VP1 of coxsackievirus A6(CVA6).Methods: The amino acid sequence of the CVA6 VP1 was analyzed using Bioedit software and various online tools including SubLoc ,TargetP and the others from ExPASy Bioinformatics Resource Portal.Results: The CVA6 VP1 protein was a hydrophilic protein with a relative molecular weight of 33.6 kD and an isoelectric point of 7.92.This protein containsed 24 phosphorylation sites , but no signal peptide , transmembrane domains and possible fatty acylation sites.Its secondary structure was characterized by the richest random coils , and 48.52 percent of its amino acid residues exposed at the solution inter-face.Epitope prediction by Bepipred showed a number of potential B cell epitopes in the protein ,the highest antigenicity index among them located in the region of amino acids residue 155-165.Conclusion:The basic physicochemical properties ,structure and function characteristics ,and potential linear B-cell epitopes of CVA 6 VP1 were successfully predicted , which laid foundations for the further study on the protein and the preparation of vaccines and immunological diagnostic reagents for CVA 6 infection.

Prediction and identification of B-cell epitopes of Treponema pallidum repeat protein F / 中国人兽共患病学报

To predict and identify the dominant B‐cell epitopes of conserved region of Treponema pallidum repeat protein F (TprFN ) and provide the basis for development of polyvalent epitope‐based syphilis vaccine ,the amino acid sequence of TprFN was obtained from GenBank and analyzed with comprehensive meta‐analysis Mobyle ,ABCpred and IEDB online software .The peptides containing predicted epitopes were artificially synthesized . To obtain and measure the titers of antibodies against TprFN ,New Zealand rabbits were immunized with recombinant protein TprFN expressed in E .coli and identified by Western blot (WB) .Sera from TprFN‐immunized rabbits ,syphilis patients ,and normal human and normal rabbits were used to deter‐mine the immunoreactivity and specificity of 7 predicted peptides of TpFN by indirect ELISA .Comprehensive meta‐analysis of online software showed that P1 (43‐62AA) ,P2(57‐71AA) ,P3(81‐88AA) ,P4(89‐103AA) ,P5(125‐138AA) ,P6(231‐251AA) and P7(268‐279AA) might be the B‐cell epitopes .A protein was expressed in a soluble form and identified as TpFN by WB .The ELISA indicated that P1 and P3 were active with TprFN‐immunized rabbit sera and syphilis patient sera but not with negative control sera .These results indicate that P1 and P3 are the potential dominant B‐cell epitopes .

Specific stimulation of B cell epitope peptides from Mycobacterium tuberculosis antigen on human peripheral γδT cells / 中国免疫学杂志

Objective:To investigate the specific stimulation of the B cell epitope peptides of Mycobacterium tuberculosis antigen (Mtb-Ag) on human peripheral γδ T cell proliferation.Methods: We selected the sequences of B cell epitope peptide from Mtb-Ag that were reported in literature and T cell epitope peptide that recently identified in this laboratory to synthesize six peptides of B cell epitopes (BP1-BP6) and two peptides of γδ T cell epitopes (TP14,TP15).The 24-well culture plates were coated with these peptides.The PBMCs were isolated from peripheral blood of healthy individuals and stained with CFSE,followed cultured for 12 days in the IL-2 containing medium.Mtb heat resistant antigen ( Mtb-HAg ) group as positive control and IL-2 only group as negative control.The percentages and proliferation index of γδ T cells were determined by flow cytometry.Results: By using Wilcoxon signed rank test for paired comparison of negative control group,the percentages of γδ T cells in cultured PBMCs with BP2,BP4 and TP14, TP15 and Mtb-HAg increased significantly in 14 samples (P<0.05);and the proliferation index of γδT cell in cultured PBMCs with BP2,BP4,BP5,BP6 and TP14,TP15 increased significantly in 7 samples (P<0.05).Conclusion: Taken together,the B cell epitope peptides from Mtb Antigens are capable of stimulating the γδ T cell proliferation specifically in vitro.Although there was individual difference inγδT cell proliferative response to B cell epitope peptides,these results strongly suggest the B cell epitope peptides also can specifically trigger the TCR ofγδT cells.

Effect of immunotherapy of recombinant chimeric epitopes of major aller-gen group 1 from Dermatophagoides farina on asthma of mice / 中国血吸虫病防治杂志

Objective To investigate the effect of immunotherapy of recombinant chimeric epitopes of major allergen group 1 from Dermatophagoides farina on asthma of mice. Methods Forty mice were randomly divided into 4 groups:a negative con?trol group,an asthma group,an immunotherapy group of Der f 1,and an immunotherapy group of Der f 1A. On the 1st,7th and 14th day,the mice in the asthma group,immunotherapy group of Der f 1,and immunotherapy group of Der f 1A were injected intraperitoneally with the extract of D. farina 3 times to sensitize;and on the 21st day,the atomized inhalation was carried out for 7 days. In the control group,phosphate buffer solution(PBS)was applied for sensitization and inhalation. In the immunother?apy groups,Der f 1 and Der f 1A were applied to carry out the specific immunotherapy respectively for 30 min before the inhala?tion. Then,the leukocytes in the bronchoalveolar lavage fluid(BALF)were numbered and the pathological sections of lung tis?sues were observed;IL?5 and IFN?γin BALF and spleen cell culture supernatants(SCCS)as well as the specific IgE,IgG2a in the sera were detected. Results Compared with the asthma group,the lung inflammation of mice in the immunotherapy groups was lightened,and the total numbers of leukocytes in BALF were significantly reduced;IL?5 was significantly reduced and IFN?γwas significantly increased in BALF and SCCS of mice in the immunotherapy groups;and the specific IgE was significantly re?duced and IgG2a was significantly increased in the sera of mice in the immunotherapy groups(all P<0.01). Conclusion The recombinant chimeric epitopes of major allergen group 1 from D. farina could effectively relieve the symptom of asthma in mice, so as to provide the evidence for specific immunotherapy.

Prediction and identification of linear B-cell epitopes in major group 3 aller-gen derived from Dermatophagoides farina / 中国血吸虫病防治杂志

Objective To predict and identify the linear B-cell epitopes in the major group 3 allergen derived from Derma-tophagoides farina(Der f 3). Methods The linear B-cell epitopes of Der f 3 allergen were analyzed based on the physicochemi-cal properties of amino acids including antigenicity,surface accessibility,flexibility,hydrophilicity,beta-turn by online bioinfor-matics softwares. The eight predicted peptides of linear B-cell epitopes were artificially synthesized and incubated with three aller-gic serum pools(4 serum samples in each),which were consisted of total 12 serum samples from the allergic individuals,and the strong positive epitopes were selected. Results Eight B-cell epitopes from Der f 3 were predicted successfully. Five of eight B-cell epitopes were identified with strong IgE-binding abilities followed by specific IgE assay. The amino acid sequences of them were following:33KAKAGDCP40, 86HASGGEKIQVAEIYQHENYDSMTID110, 118LKTPMTLDQTNAKPVPLPPQGSDVKVG144, 156QEGSYSLP163 and 199DVANGGVDSCQGDSGGPVVD218. Conclusions Five linear B-cell epitopes of Der f 3 allergen have been identified successfully. This result might provide a basis of the diagnosis and treatment for asthma.

Prediction of secondary structure and B-cell epitope of human IL-37 / 中国免疫学杂志

Objective:To predict the secondary structure and B-cell epitope of human IL-37.Methods:Based on IL-37b ami-no acid sequence, the secondary structure was predicted by SOPMA; hydrophilicity, flexibility, accessibility index were predicted by software of ProScale, Bcepred, respectively.Combined the results according to these methods , the B cell epitopes of IL-37b were pre-dicted.Results: The second structure of IL-37b contained extended strand (31.65%), random coil (52.75%), alpha helix (8.26%), beta turn (7.34%) and the most possible epitopes of IL-37b were located in or adjacent to amino acid 21-27, 34-75, 175-192 , 213-215 .Conclusion:These results will be helpful for the estimate of the epitopes and provide a theory basis for developing mon -oclonal antibodies against human IL-37.

Construction and expression of a chimeric gene with T-/B-cell epitopes of major allergen group 1 from Dermatophagoides farina / 中国血吸虫病防治杂志

Objective To construct and express a chimeric gene with T-/B-cell epitopes of the major allergen group 1 from Dermatophagoides farina(Der f 1). Methods Two chimeric genes,Der f 1A and Der f 1B,were synthesized as B1-T1-B2-T2-B3-T3-B4-T4-B5-T5-B6 and B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5 pattens. Two recombinant vectors,pET-28a(+)-Der f 1A and pET-28a(+)-Der f 1B,were constructed for prokaryotic expression. These chimeric genes were induced by 1 mmol/L IPTG (final concentration),digested with restriction enzymes and sequenced. The chimeric proteins were analyzed by SDS-PAGE and Western blotting. Results After digestion by restriction enzymes and sequencing,the recombinant vectors were constructed successfully. The specific bands for chimeric proteins were visible by SDS-PAGE and Western blotting,suggesting that these proteins were purified successfully. Further analyses were performed for IgE-binding properties of Der f 1A and Der f 1B to sera from patients sensitized to house dust mite. Compared with the parental allergens Der f 1,Der f 1A and Der f 1B had reduced IgE-binding capacity(both P0.05). Conclusion Two chimeric proteins are expressed successfully,which contain T-/B-cell epitopes of Der f 1 and provide a basis for specific immunotherapy.

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

PURPOSE: Cockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105. METHODS: Recombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining. RESULTS: The rPer a 1.0105 (~13 kDa) had 100%, 98% and > or =90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues99 QDLLLQLRDKGV110 contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 degrees Cg per gram of feces. CONCLUSIONS: The novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

PURPOSE: Cockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105. METHODS: Recombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining. RESULTS: The rPer a 1.0105 (~13 kDa) had 100%, 98% and > or =90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues99 QDLLLQLRDKGV110 contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 degrees Cg per gram of feces. CONCLUSIONS: The novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.

Cloning, sequencing of CPI gene from periodic Brugia malayi and prediction of B cell epitopes in its amino acid sequence / 中国地方病学杂志

Objective To clone and sequence the cysteine protease inhibitor gene of periodic Brugia malayi(BmCPI) and predict B-cell epitopes in amino acide sequence of BmCPI in order to provide basis for further study the expression of BmCPI and its function. Methods Total RNA was extracted from periodic Brugia malayi.A couple of specific primers were designed on the basis of known sequences of cysteine protease inhibitor gene from BmCPI. The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli(E, coli) strain DH5α. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. Five parameters and methods were used to predict B-cell epitopes in amino acide sequence of BmCPI. Results For RT-PCR, a specific band of around 621 bp was amplified. The same band was obtained by double restriction of recombinant plasmids or PCR using recombinant plasmid as template. The result of DNA sequencing showed that BmCPI shares 99% nucleotide sequence identity with that of published sequence. It showed that B-cell epitopes were probably at or adjacent to 23 - 32, 50 - 79 and 117 - 126 in its amino acide sequence. Conclusions pGEM-BmCPI is successfully constructed and sequenced, anticipated objective is reached and conditions is provided for further study of BmCPI expression and its function.

Phage display and immunological identification of efficient T- and B-combined antigenic epitopes in Helicobacter pylori adhesin A / 中华微生物学和免疫学杂志

Objective To analyze and determine the efficient T- and B-combined (T/B) antigenic epitopes in Helicobacter pylori adhesin A. Methods Recombinant HpaA (rHpaA) was expressed for immunizing rabbit to generate antiserum. T- and B-cell epitopes in HpaA molecule were predicted by using bioinformatic technique. The segments to encode T/B combined epitope peptides were amplified by PCR and the phage display systems of T/B combined epitopes were subsequently constructed. PEG/NaCl precipitation method was applied to extract the recombinant phage PⅢ (rPⅢ) that displayed T/B combined epitopes. By using either commercial IgG against whole-cell of Helicobacter pylori or rHpaA antiserum as the primary antibody, the T/B combined epitopes displayed in rP Ⅲ s were screened and identified by Western blot and ELISA. MTT was applied to determine the proliferation of rHpaA-immunized mouse splenocytes after stimulation of the different recombinant rPⅢ proteins. Results In the HpaA molecule there were five T/B combined epitopes: HpaA10, HpaA37, HpaA79, HpaA116 and HpaA143. All the T/B combined epitopes were successfully displayed on the surface of PⅢ protein of phage M13. The results of Western blot, ELISA and MTT confirmed that HpaA116 was the predominant antigenic epitope, both HpaA37 and HpaA79 were the efficient antigenic epitopes. However, HpaA10 and HpaA143 were identified as ineffective antigenic epitopes. Conclusion The phage display systems of T/B combined epitope peptides of H. pylori adhesin A have been successfully generated in this study. HpaA37 and HpaA79, especially HpaA116 are the efficient T/B combined antigenic epitopes in HpaA of H. pylori.

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.