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1.
J Med Virol ; 96(1): e29388, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38235845

RESUMO

The use of precise epitope peptides as antigens is essential for accurate serological diagnosis of viral-infected individuals, but now it remains an unsolvable problem for mapping precise B cell epitopes (BCEs) recognized by human serum. To address this challenge, we propose a novel epitope delimitation (ED) method to uncover BCEs in the delineated human IgG-reactive (HR) antigenic peptides (APs). Specifically, the method based on the rationale of similarities in humoral immune responses between mammalian species consists of a pair of elements: experimentally delineated HR-AP and rabbit-recognized (RR) BCE motif and corresponding pair of sequence alignment analysis. As a result of using the ED approach, after decoding four RR-epitomes of human papillomavirus types 16/18-E6 and E7 proteins utilizing rabbit serum against each recombinant protein and sequence alignment analysis of HR-APs and RR-BCEs, 19 fine BCEs in 17 of 22 known HR-APs were defined based on each corresponding RR-BCE motifs, including the type-specificity of each delimited BCE in homologous proteins. The test with 22 known 16/20mer HR-APs demonstrated that the ED method is effective and efficient, indicating that it can be used as an alternative method to the conventional identification of fine BCEs using overlapping 8mer peptides.


Assuntos
Proteínas Oncogênicas Virais , Peptídeos , Animais , Humanos , Coelhos , Sequência de Aminoácidos , Peptídeos/genética , Epitopos de Linfócito B , Alinhamento de Sequência , Imunoglobulina G , Mapeamento de Epitopos/métodos , Mamíferos
2.
Protein Expr Purif ; 114: 23-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26087025

RESUMO

Antibodies targeting a single epitope of the cystic fibrosis transmembrane conductance regulator (CFTR) have been reported to influence the validity of immunological analyses; however, autoimmune mechanisms associated with CFTR epitopes are not well understood. In this study, antiserum raised against a multi-epitope recombinant protein composed of three peptide fragments of CFTR (r-CFTR-3P) was prepared and B cell epitope mapping of the protein was carried out using biosynthetic peptides. The r-CFTR-3P gene was cloned into the pSY621 expression plasmid and the protein was expressed in the BL21 strain of Escherichia coli. The rabbit r-CFTR-3P antiserum recognized the native CFTR antigen extracted from human sperm and the GST188 fusion peptides CFTR(25-36), CFTR(103-117), and CFTR(1387-1480) spanning different regions of CFTR. Four novel r-CFTR-3P B cell epitopes were identified: (29)RQRLEL(34), (104)RIIASY(109), (111)PDN(113), and (1447)VKLF(1450) of CFTR. Other proteins from various species shared sequence homology with the identified epitopes based on NCBI BLAST alignment. This study provides new tools for detecting CFTR protein and insight into the characteristics of minimal B cell epitopes of CFTR and associated immunological mechanisms.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Epitopos de Linfócito B , Fragmentos de Peptídeos , Proteínas Recombinantes de Fusão , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Escherichia coli/genética , Humanos , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Espermatozoides/química
3.
Clin Dev Immunol ; 2012: 831010, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22162720

RESUMO

The human zona pellucida glycoprotein-3 (hZP3) by virtue of its critical role during fertilization has been proposed as a promising candidate antigen to develop a contraceptive vaccine. In this direction, it is imperative to map minimal motifs of the B cell epitopes (BCEs) so as to avoid ZP-specific oophoritogenic T cell epitopes (TCEs) in the ZP3-based immunogens. In this study, based on known results of mapping marmoset and bonnet monkey ZP3 (mstZP3 and bmZP3), two predictable epitopes(23-30 and 301-320) on hZP3 were first confirmed and five minimal motifs within four epitopes on hZP3 were defined using serum to recombinant hZP3a(22-176) or hZP3b(177-348) as well as a biosynthetic peptide strategy. These defined minimal motifs were QPLWLL(23-28) for hZP3(23-30), MQVTDD(103-108) for hZP3(93-110), EENW(178-181) for hZP3(172-190), as well as SNSWF(306-310) and EGP(313-315) for hZP3(301-320), respectively. Furthermore, the antigenicity of two peptides for hZP3(172-187) and hZP3(301-315) and specificity of the antibody response to these peptides were also evaluated, which produced high-titer antibodies in immunized animals that were capable of reacting to ZP on human oocytes, r-hZP3b(177-348) protein, as well as r-hZP3(172-190), r-hZP3(303-310), and r-hZP3(313-320) epitope peptides fused with truncated GST188 protein.


Assuntos
Proteínas do Ovo/imunologia , Epitopos de Linfócito B/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Zona Pelúcida/metabolismo , Animais , Proteínas do Ovo/metabolismo , Mapeamento de Epitopos , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Coelhos , Receptores de Superfície Celular/metabolismo , Zona Pelúcida/imunologia , Glicoproteínas da Zona Pelúcida
4.
Wei Sheng Yan Jiu ; 41(5): 799-804, 2012 Sep.
Artigo em Zh | MEDLINE | ID: mdl-23213697

RESUMO

OBJECTIVE: The effects of drinking boron exposure on the mass, organ indexes and structure of adrenal gland were studied in the paper. Methods 192 Sprague-Dawley rats (28 +/- 2 days) with no bacteria infecting were divided into six groups (n = 32, male = female) randomly. Treated rats drunk the distilled water which supplemented with boron of 0, 40, 80, 160, 320 and 640 mg/L, respectively, for 60 days. At the 30th and the 60th day of experiment, 16 rats (n = 8, male = female) of each group were selected and made into narcosis with 10% Chloral Hydrate. The adrenal glands were obtained, weighted and fixed after dissection, then the samples were made into paraffin sections, stained with HE stain and chromaffin, observed and photographed by Olympus CH-30 microphotograph system. RESULTS: Compared with control group, the average mass of adrenal gland of male rats in each experiment group decreased significantly or most significantly at the 30th day of experiment (P < 0.05 or P < 0.01), but the index of adrenal gland of male rats in the group of 640 mg/L boron at 60th day of experiment increased significantly (P < 0.05). Under the microscope, the microstructure of adrenal gland of rats in the group of 40 mg/L boron were better obviously than control group, and the numbers of chromaffin granules in chromaffin cell increased obviously. The histopathological changes of different degree could be observed in the group of 80 to 640 mg/L boron, and they became remarkable with the boron supplementation. By comparative observation, the damage of cells in adrenal medulla appeared ahead of them in adrenal cortex, and the pathological change of adrenal gland in male rats were obvious than female rats. CONCLUSION: Drinking supplemented with 40 mg/L boron could prompt the structure of adrenal gland in rats, but could cause different degree damage, or even obvious toxic effect when the concentration of boron supplementation in drinking from 80 to 640 mg/L.


Assuntos
Glândulas Suprarrenais/ultraestrutura , Boro/toxicidade , Água Potável/análise , Poluentes da Água/toxicidade , Animais , Boro/análise , Relação Dose-Resposta a Droga , Disruptores Endócrinos/análise , Disruptores Endócrinos/toxicidade , Feminino , Masculino , Ratos , Ratos Sprague-Dawley , Poluentes da Água/análise
5.
Hum Reprod ; 25(2): 317-27, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19923167

RESUMO

BACKGROUND: Our previous studies have demonstrated the cystic fibrosis transmembrane conductance regulator (CFTR) is important for capacitation and male fertility in mouse and guinea pig spermatozoa. However, the exact function of CFTR on human sperm fertilizing capacity, and correlation with sperm quality has not been established. The present study may shed light on some unexplained male infertility, and on a possible new method for diagnosis of male infertility and strategy for male contraception. METHODS: To assess the effect of CFTR on human sperm fertilizing capacity, we examined sperm capacitation and the acrosome reaction using chlortetracycline staining, analyzed sperm hyperactivation by computer-assisted semen analysis (CASA), measured intracellular cAMP levels using ElA and evaluated sperm penetration of zona-free hamster eggs assay in fertile men. The percentage of spermatozoa expressing CFTR from fertile, healthy and infertile men (mainly teratospermic, asthenoteratospermic, asthenospermic and oligospermic) was conducted by indirect immunofluorescence staining. RESULTS: Progesterone significantly facilitated human sperm capacitation and ZP3 triggered the acrosome reaction, both were significantly inhibited by CFTR inhibitor-172 (CFTRinh-172; 10 nM-1 microM) in a dose-dependent manner. The presence of 100 nM CFTRinh-172 markedly depressed intracellular cAMP levels, sperm hyperactivation and sperm penetration of zona-free hamster eggs. In addition, the percentage of spermatozoa expressing CFTR in the fertile men was significantly higher than healthy and infertile men categories (P < 0.01). CONCLUSIONS: CFTR is essential for human sperm fertilizing capacity and the impairment of CFTR expression in spermatozoa is correlated with a reduction of sperm quality. These results suggest that defective expression of CFTR in human sperm may lead to the reduction of sperm fertilizing capacity.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Infertilidade Masculina/fisiopatologia , Capacitação Espermática/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/metabolismo , Reação Acrossômica/efeitos dos fármacos , Adulto , Animais , Benzoatos/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Expressão Gênica , Humanos , Masculino , Progesterona/antagonistas & inibidores , Progesterona/farmacologia , Análise do Sêmen , Motilidade dos Espermatozoides/efeitos dos fármacos , Tiazolidinas/farmacologia
6.
PLoS One ; 14(10): e0223978, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618247

RESUMO

Guertu virus (GTV) is a tick-borne phleboviruses (TBPVs) which belongs to the genus Banyangvirus in the family of Phenuiviridae. In vitro and in vivo studies of GTV demonstrated that it was able to infect animal and human cell lines and could cause pathological lesions in mice. Glycoproteins (GP, including Gn and Gc) on the surface of Guertu virus (GTV) could bind to receptors on host cells and induce protective immunity in the host, but knowledge is now lacking on the information of B cell epitopes (BCEs) present on GTV-GP protein. The aim of this study was to identify all BCEs on Gn of the GTV DXM strain using rabbit pAbs against GTV-Gn. Seven fine BCEs and two antigenic peptides (APs) from nine reactive 16mer-peptides were identified, which are EGn1 (2PIICEGLTHS11), EGn2 (135CSQDSGT141), EGn3 (165IP EDVF170), EGn4 (169VFQEL K174), EGn5 (187IDGILFN193), EGn6 (223QTKWIQ228), EGn7 (237CHKDGIGPC245), AP-8 (299GVRVRPKCYGFSRMMA314) and AP-9 (355CASH FCSSAESGKKNT370), of which six of mapped BCEs were recognized by the IgG-positive sheep serum obtained from sheep GTV-infected naturally. Multiple sequence alignments (MSA) based on each mapped BCE motif identified that the most of identified BCEs and APs are highly conserved among 10 SFTSV strains from different countries and lineages that share relatively close evolutionary relationships with GTV. The fine epitope mapping of the GTV-Gn would provide basic data with which to explore the GTV-Gn antigen structure and pathogenic mechanisms, and it could lay the foundation for the design and development of a GTV multi-epitope peptide vaccine and detection antigen.


Assuntos
Mapeamento de Epitopos/métodos , Glicoproteínas/química , Peptídeos/metabolismo , Phlebovirus/metabolismo , Sequência de Aminoácidos , Animais , Modelos Moleculares , Conformação Proteica , Coelhos , Alinhamento de Sequência , Ovinos/imunologia , Proteínas do Envelope Viral/química
7.
PLoS One ; 13(9): e0204264, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30235312

RESUMO

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne pathogen that causes severe disease in humans. CCHFV is widely distributed in more than 30 countries and distinct regions, which means that it poses a serious threat to human health. The nucleocapsid protein (NP) encoded by the CCHFV S gene is the primary detectable antigen in infected cells, which makes it an important viral antigen and a clinical diagnostic target. In this study, the modified biosynthetic peptide (BSP) method was used to identify the fine epitopes on the N- and C- terminals of NP from the CCHFV YL04057 strain using rabbit antiserum against CCHFV-NP. Nine epitopes were identified: E1a (178NLILNRGG185), E1b (184GGDENP189), E2 (352PLKWGKK358), E3 (363FADDS367), E4 (399NPDDAA404), E5a (447DIVASEHL454), E5b (452EHLLHQSL459), E6 (464SPFQNAY470) and E7 (475NATSANII482). Western blotting analysis showed that each epitope interacted with the positive serum of sheep that had been naturally infected with CCHFV. Amino acid sequence alignment between each epitope and their homologous proteins showed that they were almost 100% conserved among 12 CCHFV sequences from different lineages, except for epitopes E1a, E1b and E2. Three-dimensional structural modeling analysis showed that all identified epitopes were located on the surface of the NP "head" domain. This study identified fine epitopes on the N- and C- terminals of NP, which will increase the understanding of the structure and function of NP, and it could lay the foundation for the design and development of a CCHFV multi-epitope peptide vaccine and detection antigen.


Assuntos
Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Proteínas do Nucleocapsídeo/química , Animais , Anticorpos Antivirais/metabolismo , Sequência Conservada , Epitopos de Linfócito B/química , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Modelos Moleculares , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/imunologia , Domínios Proteicos , Coelhos
8.
Artigo em Inglês | MEDLINE | ID: mdl-30290884

RESUMO

Glycoprotein (GP) is a major antigen of Crimean-Congo hemorrhagic fever virus (CCHFV), and binds to its receptor on the host cell and induces protective immunity in the host. The aim of this study is to identify all linear B cell epitopes (BCEs) on the N-terminal glycoprotein (Gn) of CCHFV using a modified overlapping peptide biosynthesis method. The eight fine BCEs (Gn-E1a, 543RTQLV547; E1b, 553EIH555; E1c, 554IHEDSY559; E1d, 557DSYG560; E2, 615CKQGFC620; E3a, 657GDILVD662; E3b, 662DCSGGQQH669, and E4, 678LGCPNVPL685) were identified using the rabbit antisera, which all were recognized by serum from IgG-positive sheep CCHFV-infected naturally in Western blotting. The multiple sequence alignment (MSA) revealed high conservation of the identified BCEs among ten CCHFV strains from different areas. These mapped epitopes of the CCHFV Gn would provide a basis for further the elucidation of CCHFV pathogenesis, and the development of CCHFV multi-epitope vaccines and detection reagents.


Assuntos
Epitopos de Linfócito B/imunologia , Glicoproteínas/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Mapeamento de Epitopos/métodos , Imunoglobulina G/imunologia , Coelhos , Alinhamento de Sequência , Ovinos , Vacinas/imunologia
9.
J Androl ; 28(3): 381-8, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17192598

RESUMO

To explore the biological characteristics of the recombinant zona pellucida 3 (ZP3) peptides of rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348, we examined whether rhuZP3a22 approximately 176 or rhuZP3b177 approximately 348 trigger the acrosome reaction (AR) of human spermatozoa and we investigated the underlying mechanism. The assessment of AR was performed using chlortetracycline staining. The intracellular free calcium concentration ([Ca2+]i) in Fura-2/AM-loaded human sperm was monitored with a spectrofluorophotometer. We found that rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348 were capable of eliciting AR at different concentrations. With the addition of either peptide, the [Ca2+]i level was raised to a peak with or without a plateau. The AR could be inhibited by pertussis toxin (PTX), EGTA, and pimozide (a T-type calcium channel blocker), whereas verapamil was less effective in this regard. The results of the present study suggest that peptides rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348 have a role similar to human ZP3, and that the mechanism of the response to the peptides involves influx of calcium, the G protein pathway, and a T-type calcium channel.


Assuntos
Reação Acrossômica/fisiologia , Cálcio/metabolismo , Proteínas do Ovo/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Espermatozoides/fisiologia , Ácido Egtázico , Humanos , Masculino , Peptídeos/fisiologia , Toxina Pertussis , Pimozida , Proteínas Recombinantes , Espermatozoides/metabolismo , Glicoproteínas da Zona Pelúcida
10.
PLoS One ; 12(10): e0186097, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29023483

RESUMO

There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.


Assuntos
Mapeamento de Epitopos/métodos , Glutationa Transferase/metabolismo , Peptídeos/metabolismo , Plasmídeos/genética , Engenharia de Proteínas/métodos , Animais , Anticorpos Monoclonais/metabolismo , Mapeamento de Epitopos/economia , Glutationa Transferase/genética , Imunização , Masculino , Proteínas Oncogênicas Virais/genética , Peptídeos/imunologia , Engenharia de Proteínas/economia , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
11.
Am J Reprod Immunol ; 75(4): 474-85, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26782177

RESUMO

PROBLEM: The development of a new and suitable contraceptive methods, as well as in-depth and systematic research into underlying contraceptive mechanisms, is crucial. IZUMO1 plays an important role in the fusion of the sperm and ovum during fertilization. Izumo(-/-) mice are infertile. Therefore, IZUMO1 may be a potential target for the development of a contraceptive vaccine. METHOD OF STUDY: Linear B-cell epitopes (BCE) were identified in IZUMO using biosynthetic peptides and used to immunize female mice. RESULTS: Five IZUMO BCE were identified: DLVLDCL177-183, YSFYRV196-201 (named BCE-2), YLT217-219, SMVGPED221-227, and DAGNY228-232. Active immunization with the BCE-2 vaccine sharply decreased the fertility rate in female mice in a safe and reversible manner. In vitro fertilization showed that the BCE-2 vaccine interferes with and blocks the fusion of the sperm and the ovum. CONCLUSIONS: B-cell epitopes-2 may be a new candidate for the development of contraceptive vaccine due to its effectiveness, safety, and reversibility.


Assuntos
Anticoncepção Imunológica/métodos , Epitopos de Linfócito B/farmacologia , Imunoglobulinas/farmacologia , Proteínas de Membrana/farmacologia , Peptídeos/farmacologia , Vacinas Anticoncepcionais/farmacologia , Animais , Epitopos de Linfócito B/imunologia , Feminino , Imunoglobulinas/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/imunologia , Vacinas Anticoncepcionais/imunologia
12.
Sci Rep ; 6: 34686, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27708433

RESUMO

To enable rational multi-epitope vaccine and diagnostic antigen design, it is imperative to delineate complete IgG-epitome of the protein. Here, we describe results of IgG-epitome decoding of three proteins from high-risk (HR-) oncogenic human papillomavirus type 58 (HPV58). To reveal their entire epitomes, employing peptide biosynthetic approach, 30 precise linear B-cell epitopes (BCEs) were mapped on E6, E7 and L1 proteins using rabbits antisera to the respective recombinant proteins. Using sequence alignment based on BCE minimal motif, the specificity and conservativeness of each mapped BCE were delineated mainly among known HR-HPVs, including finding 3 broadly antibody cross-reactive BCEs of L1 that each covers almost all HR-HPVs. Western blots revealed that 13 of the 18 BCEs within L1-epitome were recognized by murine antisera to HPV58 virus-like particles, suggesting that these are antibody accessible BCEs. Also, a highly conserved epitope (YGD/XTL) of E6 was found to exist only in known common HR-HPVs, which could be used as the first peptide reference marker for judging HR-HPVs. Altogether, this study provides systemic and exhaustive information on linear BCEs of HR-HPV58 that will facilitate development of novel multi-epitope diagnostic reagents/chips for testing viral antibodies and 'universal' preventive HPV peptide vaccine based on L1 conserved BCEs.


Assuntos
Proteínas do Capsídeo/química , Mapeamento de Epitopos/métodos , Imunoglobulina G/sangue , Proteínas Oncogênicas Virais/química , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/química , Animais , Sítios de Ligação , Proteínas do Capsídeo/imunologia , Epitopos de Linfócito B/análise , Humanos , Camundongos , Modelos Moleculares , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Conformação Proteica , Coelhos
13.
Sheng Li Xue Bao ; 57(6): 682-8, 2005 Dec 25.
Artigo em Zh | MEDLINE | ID: mdl-16344891

RESUMO

The present study was aimed to analyze the immunogenicity of recombinant human zona pellucida-3 peptides (r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348)) expressed in E. coli through immunizing rabbits, and to evaluate the efficacy of their polyclonal antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding respectively. Male New Zealand rabbits were immunized using r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) as antigen respectively, which was purified through an improved method of preparative gel polyacryulamide gel electrophoresis. The antibody response level of r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) immunogen in rabbits was determined by ELISA using mouse ZP3-5 (amino acid sequence(137 approximately 150) being completely conserved with huZP3(138 approximately 151) sequence) and specific huZP3-14 (amino acid sequence(327 approximately 340)) synthetic peptides as coating antigens respectively. The immunoreactivity and specificity of the anti-r-huZP3a(22 approximately 176) and anti-r-huZP3b(177 approximately 348) antisera with each r-huZP3 peptides, were tested by immunoblot and immunohistochemistry (using native huZP and human ovary section) respectively. A competitive hemizona assay (HZA) was used to evaluate the efficacy of the antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding. Both r-huZP3 peptides were able to induce higher antibody titers in rabbits. Each antiserum could specifically recognize or bind to each target r-huZP3 peptide expressed in E. coli and native human ZP in vitro. The antisera also inhibited sperm-egg binding in the HZA. These results show that r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) are of strong immunogenicity. They can be used to develop a kit for detecting whether there are autoantibodies to zona pellucida in unexplained infertile women, and their antisera might be useful tools for determining minimal B-cell epitope sequences of several known huZP3 epitope peptides.


Assuntos
Proteínas do Ovo/biossíntese , Proteínas do Ovo/imunologia , Soros Imunes/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes/imunologia , Interações Espermatozoide-Óvulo/imunologia , Animais , Proteínas do Ovo/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Imunização , Masculino , Glicoproteínas de Membrana/genética , Coelhos , Receptores de Superfície Celular/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Glicoproteínas da Zona Pelúcida
14.
Artigo em Zh | MEDLINE | ID: mdl-26248424

RESUMO

OBJECTIVE: To establish and evaluate a BA-ELISA method for the quantitative detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein. METHODS: We deliberately selected three tables of CFTR and made the synthetic peptide be expressed in E. coli, then used the antigen to immunize rabbits to obtain the anti-CFTR polyclonal serum. After that, 96 well plates were coated with the purified antibody against CFTR. The antigen CFTR which was extracted from human sperm was detected by anti-CFTR antibody labeled with biotin, horseradish peroxidase conjugated avidin, and the substrate. The concentrations of two kinds of antibodies and the experiment parameters were optimized. Thereby, the double antibody sandwich BA-ELISA method for the quantitative detection of CFTR protein was established. Furthermore, the reproducibility, specificity and so on were evaluated by clinical specimens of sperm. RESULTS: The optimal concentration of coated anti-CFTR IgG was 4 µg/ml, while the biotin labeled anti-CFTR IgG was 10 µg/ml; the optimal blocking buffer was 1% BSA-PBST, the optimal time of the reaction between antigen and antibody was 60 min, the optimal chromogenic time was 15 min, the intra-assay and inter-assay coefficient were 2.16%-9.23% and 2.29%-11.71% respectively; The lowest detectable limit was 0.15 ng/ml; the standard curve had a good linear correlation of R2 = 0.962. CONCLUSION: The BA-ELISA method for the quantitative detection of CTFR protein is successfully established, and it is demonstrated that the method has strong specificity, high sensitivity and good reproducibility. It provides the basis and evidence of the further application of the method.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/análise , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Anticorpos , Escherichia coli , Humanos , Peptídeos , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
15.
Vet Microbiol ; 175(1): 132-8, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25465659

RESUMO

Nucleoprotein (NP) is the most abundant and highly immunogenic protein of morbillivirus, and is presently the basis of most diagnostic assays for peste des petits ruminants virus (PPRV). In this study, fine epitope mapping and conservation analysis of linear B-cell epitopes on the PPRV NP has been undertaken using biosynthetic peptides. Nineteen linear B-cell epitopes were identified and their corresponding minimal motifs were located on the NP of PPRV China/Tibet/Geg/07-30. Conservation analysis indicated that ten of the 19 minimal motifs were conserved among 46 PPRV strains. Peptides containing the minimal motifs were recognized using anti-PPRV serum from a goat immunized with PPRV vaccine strain Nigeria 75/1. Identified epitopes and their motifs improve our understanding of the antigenic characteristics of PPRV NP and provide a basis for the development of epitope-based diagnostic assays.


Assuntos
Epitopos de Linfócito B/imunologia , Doenças das Cabras/prevenção & controle , Cabras/imunologia , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Vacinação/veterinária , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Mapeamento de Epitopos/veterinária , Feminino , Doenças das Cabras/imunologia , Doenças das Cabras/virologia , Dados de Sequência Molecular , Nucleoproteínas/imunologia , Peste dos Pequenos Ruminantes/diagnóstico , Coelhos , Proteínas Recombinantes , Proteínas Virais/imunologia
16.
Artigo em Zh | MEDLINE | ID: mdl-12198575

RESUMO

In recent years, development of chimeric peptide (CP) immunogens is a trend in the vaccinological field. The CPs contain a B cell epitope(s) of target antigen and a promiscuous self - or foreign- T cell epitope(s). However, such constructed CPs were all expressed in prokaryotic or eukaryotic systems at lower levels. To purify the human chorionic gonadotropin (hCG) CP12 expressed in E. coli at the level of about 1% of the total cell proteins, an improved method of preparative gel polyacrylamide gel electrophoresis (PAGE) was developed. The important improvement to routine preparative PAGE involves: (1) running reversed electrophoresis by rearranging the gel- carrying plate when the bromophenol blue band arrived at 1-1.5 centimeter from the bottom of the gel; (2) making a collecting trough between the gel and a dialytic membrane that was used to isolate the upper tank buffer. About 8 fractions were collected at regular intervals of 15 minutes after bromophenol blue running out of gel. And then 0.2 ml was taken from each fraction and the protein was precipitated by sequentially adding trichloroacetic acid and acetone. Each sample was dissolved in 20 microL sample buffer and analyzed and identified by SDS-PAGE and Western blotting. As a result, the hCG CP12 expression product with 95% relative homogeneity was harvested at a 50-100 microgram level after a single-step purification of this preparative PAGE, with respect to the sample which contained 3-4 mg of cell proteins.


Assuntos
Gonadotropina Coriônica/genética , Expressão Gênica , Western Blotting/métodos , Gonadotropina Coriônica/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
17.
Am J Reprod Immunol ; 68(6): 465-75, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22860757

RESUMO

PROBLEM: To decipher structural and functional aspects of human zona pellucida glycoprotein-4 (ZP4), the epitopes recognized by monoclonal antibodies (MAbs) have been mapped. METHOD OF STUDY: Recombinant human ZP4-mediated induction of acrosome reaction in human sperm was studied in the absence and presence of ZP4-specific MAbs. The epitopes of MAbs were mapped using recombinant peptides expressed in Escherichia coli. RESULTS: Monoclonal antibodies (MA-1662, MA-1671) against human ZP4 showed specific binding to ZP matrix of human eggs in an indirect immunofluorescence assay. Both the antibodies showed significant (P < 0.05) inhibition in the baculovirus-expressed recombinant ZP4-mediated acrosome reaction. MA-1671 recognized N-terminal fragment of ZP4 and minimal epitope mapped to amino acid residues 126-130 (PARDR), whereas MA-1662 reacted to C-terminal fragment and minimal epitope mapped to amino acid residues 256-260 (ENELV). CONCLUSIONS: The epitopes corresponding to both N- and C-terminal parts of human ZP4 may be relevant for its biological activity.


Assuntos
Reação Acrossômica , Proteínas do Ovo/imunologia , Proteínas do Ovo/fisiologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/fisiologia , Zona Pelúcida/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Proteínas do Ovo/química , Mapeamento de Epitopos , Humanos , Masculino , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Proteínas Recombinantes , Alinhamento de Sequência , Espermatozoides/imunologia , Glicoproteínas da Zona Pelúcida
18.
J Biotechnol ; 151(1): 15-21, 2011 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-21084058

RESUMO

To develop a superior chimeric peptide (CP) vaccine of human chorionic gonadotropin (hCG), two CP antigens (named CP12 and CP22) encoding one or two copies of three linear B cell epitopes from the ß-hCG subunit and six foreign T cell epitopes, including two promiscuous TCEs from hepatitis B surface antigen and tetanus toxoid, were constructed and biosynthesized. The hCG CP12 and CP22 of 21 or 23 kDa, respectively, were expressed in Escherichia coli at the level of ~1% of total cell proteins when inserted into thermo-inducible pBV221 expression vector. The purified CP12 and CP22 proteins with >95% relative homogeneity are immunogenic, and elicited antibodies against the ß5, ß9 and ß8 BCEs of ß-hCG in both rabbits and three different inbred strains of mice. A mouse uterine weight study in Balb/c mice demonstrated that the CP12 and CP22 antigens with an additional ß5 neutralizing epitope enhanced the in vivo bio-neutralization capacity of the induced antibodies compared to the C-terminal immunogen of ß-hCG. We propose that the biosynthesized CP22, possessing with two copies of three BCEs, represents a novel candidate antigen for an hCG contraceptive or tumor therapeutic vaccine.


Assuntos
Gonadotropina Coriônica Humana Subunidade beta/imunologia , Epitopos de Linfócito B/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Western Blotting , Gonadotropina Coriônica Humana Subunidade beta/química , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Eletroforese em Gel de Poliacrilamida , Epitopos de Linfócito B/química , Epitopos de Linfócito B/metabolismo , Escherichia coli , Feminino , Camundongos , Tamanho do Órgão , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e Especificidade , Estereoisomerismo , Útero/metabolismo , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/metabolismo , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismo
19.
J Reprod Immunol ; 81(1): 9-16, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19539378

RESUMO

An important step in the development of a human zona pellucida (huZP) peptide vaccine is to define the minimal amino acid motif for a mapped B cell epitope peptide within huZP4. Identification of this minimal motif is necessary to remove an overlapping T cell epitope that induces a pathogenic T cell response. Here we describe motif (PLTLEL(314-319)) mapping of an 18mer B cell epitope peptide(308-325) on huZP4 protein (previously known as huZP1/ZPB protein), achieved using a set of 22 biosynthetic 8mer peptides fused with truncated glutathione S-transferase (GST) or truncated streptavidin protein, and detected using rabbit anti-porcine zona pellucida (pZP) IgG. The immunogenicity of the B cell epitope peptide was evaluated in rabbits using expressed B cell epitope peptide fused with truncated streptavidin as the antigen. This construct elicited high titer antibody to the 18mer B cell epitope peptide, with reactivity to native human ZP, the biosynthetic 18mer peptide and the 18mer peptide GST fusion protein.


Assuntos
Motivos de Aminoácidos , Proteínas do Ovo/metabolismo , Epitopos de Linfócito B/imunologia , Glicoproteínas de Membrana/metabolismo , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Proteínas do Ovo/genética , Proteínas do Ovo/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/imunologia , Feminino , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Gravidez , Engenharia de Proteínas , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estreptavidina/genética , Estreptavidina/metabolismo , Suínos , Glicoproteínas da Zona Pelúcida
20.
Sheng Wu Gong Cheng Xue Bao ; 20(1): 49-53, 2004 Jan.
Artigo em Zh | MEDLINE | ID: mdl-16108489

RESUMO

The possibility of using a subunit or fragment of human chorionic gonadotropin (hCG) as an immunogen for birth control has been actively explored for many years. This protein homone is produced by the fertilized egg and is required for implantation of the blastocyst into the maternal uterus and the maitenance of pregnancy. In previous studies, several bio-synthesized hCG chimeric peptides (CP) that contain three linear B-cell epitopes (beta5, beta9 and beta8) of beta-hCG subunit together with various foreign 'promiscuous' T-cell epitopes were constructed and expressed as potential new hCG vaccine immunogens. In order to detect antibodies to each of the individual B-cell epitopes present in the animal antiserum raised against the hCG CPs, we decided to construct three recombinant proteins, each contains a single target B-cell epitope (betaE) of beta-hCG. Two sets of DNA fragments were chemically synthesized encoding the beta5, beta9 and beta8 epitopes (betaE) 45 approximately 52, 113 approximately 116 or 133 approximately 144 of beta-hCG subunit and were inserted into the downstream of streptavidin (Stv) gene in pTSA18 separately, with or without an extra TAA codon at the 3'-terminals of the genes. SDS-PAGE analysis revealed that only Stv-betaE (-beta5, -beta9 or -beta8) fusion genes set with the TAA codon can be expressed in E. coli BL21 (DE3) pLysS strain at high level after 1mM IPTG induction for 4 hours. Additionally, these fusion proteins can all be recognized by specific polyclonal antiserum (RS-4157) generated upon immunization with the loop peptide 38 approximately 57 of beta-hCG, monoclonal antibody (mAb) FB12 to beta9 epitope and mAb OT3A that specially recognizes reporter sequence 133 approximately 139 of beta8 epitope 137 approximately 144. Each of the proteins can be purified to 95% relative homogeneity using an improved method of preparative gel polyacrylamide gel electrophoresis. The yields were 5 mg per 1 L culture. The three target Stv-betaE fusion proteins will be useful in determining the immunogenicity of designed hCG CPs and hCG vaccines, including hCG DNA vaccines.


Assuntos
Gonadotropina Coriônica Humana Subunidade beta/imunologia , Epitopos de Linfócito B/genética , Proteínas Recombinantes de Fusão/biossíntese , Gonadotropina Coriônica Humana Subunidade beta/genética , Humanos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Estreptavidina/genética , Vacinas Sintéticas/imunologia
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