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1.
Nat Commun ; 14(1): 6601, 2023 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-37857610

RESUMO

Immunogenomic loci remain poorly understood because of their genetic complexity and size. Here, we report the de novo assembly of a cattle genome and provide a detailed annotation of the immunogenomic loci. The assembled genome contains 143 contigs (N50 ~ 74.0 Mb). In contrast to the current reference genome (ARS-UCD1.2), 156 gaps are closed and 467 scaffolds are located in our assembly. Importantly, the immunogenomic regions, including three immunoglobulin (IG) loci, four T-cell receptor (TR) loci, and the major histocompatibility complex (MHC) locus, are seamlessly assembled and precisely annotated. With the characterization of 258 IG genes and 657 TR genes distributed across seven genomic loci, we present a detailed depiction of immune gene diversity in cattle. Moreover, the MHC gene structures are integrally revealed with properly phased haplotypes. Together, our work describes a more complete cattle genome, and provides a comprehensive view of its complex immune-genome.


Assuntos
Genoma , Genômica , Bovinos , Animais , Genoma/genética , Complexo Principal de Histocompatibilidade/genética , Imunoglobulinas , Genes de Imunoglobulinas
2.
Artigo em Chinês | MEDLINE | ID: mdl-35634675

RESUMO

Objective: To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that can sensitively detect the second messenger cyclic AMP (cAMP) in the cytoplasm. Methods: The eukaryotic expression vectors of CFTR and YFP-H148Q / I152L were constructed respectively. FRT cells co-expressing CFTR and YFP-H148Q / I152L were obtained by liposome transfection. The expression of CFTR and YFP-H148Q / I152L in FRT cells was observed by an inverted fluorescence microscopy, and flow cytometry was used to detect the purity of cells; The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments. The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator. Results: The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q / I152L was expressed in the cytoplasm of FRT cells. The FRT cell model stably co-expressing ANO1 and YFP-H148Q / I152L was successfully constructed. The model could screen CFTR modulators, and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm. The cell model could sensitively detect the intracellular cAMP concentration. Conclusion: The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm, and it provided a simple and efficient method for the study of other targets associated cAMP signal.


Assuntos
AMP Cíclico , Regulador de Condutância Transmembrana em Fibrose Cística , Citoplasma , Sistemas do Segundo Mensageiro
3.
Exp Ther Med ; 21(2): 136, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33456503

RESUMO

e23sFv is a HER2-targeted single-chain variable fragment (scFV) that was characterized as the targeting portion of a HER2-targeted tumour proapoptotic molecule in our previous study. In vitro antibody affinity maturation is a method to enhance antibody affinity either by complementarity-determining region (CDR) mutagenesis or by framework region (FR) engraftment. In the present study, the affinity of e23sFv was enhanced using two strategies. In one approach, site-directed mutations were introduced into the FRs of e23sFv (designated EMEY), and in the other approach e23sFv FRs were substituted with FRs from the most homologous screened antibodies (designated EX1 and EX2). Notably, EX1 derived from the FR engraftment strategy demonstrated a 4-fold higher affinity for HER2 compared with e23sFv and was internalized into HER2-overexpressing cells; however, EMEY and EX2 exhibited reduced affinity for HER2 and decreased internalization potential compared with EX1. The 3D structure of EX1 and the HER2-EX1 complex was acquired using molecular homology modelling and docking and the HER2 epitopes of EX1 and the molecular interaction energy of the EX1-HER2 complex were predicted. In the present study, it was demonstrated that scFv affinity improvement based on sequence alignment was feasible and effective. Moreover, the FR grafting strategy was indicated to be more effective and simple compared with site-directed mutagenesis to improve e23sFv affinity. In conclusion, it was indicated that the affinity-improved candidate EX1 may present a great potential for the diagnosis and treatment of HER2-overexpressing tumours.

4.
Biomaterials ; 178: 170-182, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29935385

RESUMO

As HER2 is frequently overexpressed in various malignancies, targeting HER2 is considered an efficient, highly selective antitumor therapy. HER2-targeted immunoconjugates are being developed and result in persistent remission of HER2-overexpressing tumors. However, many of the antibodies used as the targeting moiety are of murine origin and exhibit risk of inducing immunogenicity, limiting their antitumor therapeutic efficacy. Here, we humanized e23sFv, an HER2-targeting murine scFv with excellent affinity and specificity, using a human antibody consensus sequence engraftment strategy. The affinity of the initially humanized e23sFv was then rescued and improved by selective mutagenesis followed by phage-display-based affinity panning of the mutant pool. The resulting humanized e23sFv candidates (husFvs) exhibited up-to-94-fold increased affinity to recombinant HER2. The immunogenicity of e23sFv was dramatically alleviated after humanization, as indicated by the impaired production of cytokines by husFv-stimulated human PBMCs. Two internalizable husFvs with optimal affinity were applied to generate humanized immunoapoptotins by infusion with the translocation domain Fdt and the proapoptotic domain truncated Bid. The husFv-immunoapoptotins demonstrated improved HER2-targeting and tumor-killing capacities in vitro and in vivo compared with the e23sFv-immunoapoptotins and would enable the administration of multiple treatment cycles to patients, resulting in improved antitumor efficacy. Furthermore, the husFvs recognized distinct HER2 epitopes and could thus be used in combination with trastuzumab or pertuzumab to achieve robust synergistic antitumor effects in HER2-positive malignancies.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Neoplasias/patologia , Receptor ErbB-2/imunologia , Anticorpos de Cadeia Única/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Citocinas/metabolismo , Citotoxicidade Imunológica , Endocitose , Humanos , Fragmentos de Imunoglobulinas/química , Cinética , Leucócitos Mononucleares/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação/genética , Reprodutibilidade dos Testes , Anticorpos de Cadeia Única/química
5.
Nat Microbiol ; 3(5): 622-631, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29662128

RESUMO

Insulin resistance is a risk factor for obesity and diabetes and predisposes individuals to Staphylococcus aureus colonization; however, the contribution of S. aureus to insulin resistance remains unclear. Here, we show that S. aureus infection causes impaired glucose tolerance via secretion of an insulin-binding protein extracellular domain of LtaS, eLtaS, which blocks insulin-mediated glucose uptake. Notably, eLtaS transgenic mice (eLtaS trans ) exhibited a metabolic syndrome similar to that observed in patients, including increased food and water consumption, impaired glucose tolerance and decreased hepatic glycogen synthesis. Furthermore, transgenic mice showed significant metabolic differences compared to their wild-type counterparts, particularly for the early insulin resistance marker α-hydroxybutyrate. We subsequently developed a full human monoclonal antibody against eLtaS that blocked the interaction between eLtaS and insulin, which effectively restored glucose tolerance in eLtaS trans and S. aureus-challenged mice. Thus, our results reveal a mechanism for S. aureus-induced insulin resistance.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Resistência à Insulina , Insulina/metabolismo , Infecções Estafilocócicas/complicações , Staphylococcus aureus/patogenicidade , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Linhagem Celular , Feminino , Células Hep G2 , Humanos , Hidroxibutiratos/metabolismo , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Transgênicos , Ratos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo
6.
Cancer Res ; 76(9): 2698-709, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26984756

RESUMO

All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diterpenos/farmacologia , Leucemia Mieloide/patologia , Fitoterapia/métodos , Animais , Western Blotting , Ativação Enzimática/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Zhongguo Zhong Yao Za Zhi ; 39(21): 4143-8, 2014 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-25775783

RESUMO

Suitable pretreatment of biological samples can truly reflect the role of law of the measured components played in the body and will provide experimental evidence for the studies on metabolic process, material basis of efficacy, mechanism of action, pharmacology, toxicology and the others. Biological samples include blood, urine, hair, tears, etc. There are also many samples processing methods, such as the direct protein precipitation, liquid-liquid extraction and solid phase extraction and so on. These methods could be used alone or combined.


Assuntos
Líquidos Corporais/química , Técnicas de Química Analítica/métodos , Animais , Precipitação Química , Humanos , Extração Líquido-Líquido , Proteínas/isolamento & purificação , Extração em Fase Sólida
8.
Cell Biochem Biophys ; 66(3): 753-7, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23420525

RESUMO

Extracellular fibrinogen-binding protein (Efb) of Staphylococcus aureus (S. aureus) is a bi-functional protein, which can specifically bind fibrinogen with its N terminus and inhibit deposition of C3b on the surface of S. aureus with its C terminus. Here, we screened the epitopes of Efb using phage display. Four peptides with consensus motif were screened. This consensus motif was identical to C terminus (161-164) of Efb. In the further investigation, it was found the synthesized peptide EC1 (154-165aa of Efb) could specifically bind C3/C3b and subsequently to block the activation of complement. Meanwhile, EC1 could inhibit the interaction between Efb and C3/C3b. Moreover, the interaction between the mutant protein of EmC1 (Efb without EC1) and C3 was decreased. And, the effect on the complement system of the mutant protein was dramatically declined compared with Efb. Our finding suggested that the peptide EC1 could mimic Efb to block complement system activation via binding C3.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Inativadoras do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Espaço Extracelular , Fibrinogênio/metabolismo , Mimetismo Molecular , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Morte Celular/efeitos dos fármacos , Proteínas Inativadoras do Complemento/química , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Humanos , Oligopeptídeos/química , Staphylococcus aureus
9.
Biotechnol Lett ; 35(4): 523-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23242501

RESUMO

B lymphocyte stimulator (BLyS) antagonists are new therapeutic reagents for treating the autoimmune diseases. Peptibodies can inhibit the bioactivity of BLyS, the same as other BLyS antagonists: decoyed BLyS receptors and anti-BLyS antibodies. In this study, a new optimized BLyS antagonist peptide was designed according to our previous work by the computer-aided homology modeling. Competitive ELISA showed that the peptide at 100 µg/ml could inhibit 54 % of the BCMA-Fc binding to BLyS. To maintain its stability and spatial conformation, the peptide was fused to human IgG1 Fc to form a peptide-Fc fusion protein-a novel peptibody by gene engineering. ELISA indicated that the peptibody could bind with BLyS in dosage-dependent manner as BCMA-Fc did. This study highlights the possibility of designing and optimizing BLyS antagonist peptides with high biopotency by the computer-aided design. Thus, these peptides could neutralize BLyS activity and be potential antagonists to treat autoimmune diseases related with BLyS overexpression.


Assuntos
Fator Ativador de Células B/antagonistas & inibidores , Fator Ativador de Células B/metabolismo , Antígeno de Maturação de Linfócitos B/metabolismo , Fatores Imunológicos/metabolismo , Peptídeos/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Fatores Imunológicos/genética , Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
10.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 18(6): 1585-9, 2010 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-21176375

RESUMO

This study was aimed to explore the influence of excessive complement activation on the pathological process of acute graft-versus-host disease (aGVHD) in mice. A murine model with aGVHD was established by injecting cell mixture containing splenocytes and bone marrow cells at 2:1 ratio from donor C57BL/6(H-2K(b)) mice into recipient BALB/c (H-2K(d)) mice within 4-6 hours after 8 Gy (60)Co γ-ray total body irradiation. The mice received syngeneic bone marrow transplantation were used as control group. After transplantation, the mice were monitored daily for body weight and mortality. At day 14, all mice were sacrificed and each liver was freshly dissociated for histological analysis. The hepatic mRNA abundance for complement components C3a and C5a as well as receptors for these two anaphylatoxin were tested by real-time quantitative PCR method. And the levels of C3a and C5a production in liver were detected by ELISA. The deposition of complement C3 in liver was determined by immunofluorescence staining using frozen section. The results indicated that as compared with syngeneic bone-marrow transplantation control group, experimental animals underwent aGVHD characterized by weight loss, depilation, diarrhea and lassitude. Interestingly, the hepatic mRNA expression for complement anaphylatoxin family member C3a and C5a as well as their receptors C3aR and C5aR1 in mice with aGVHD were significantly up-regulated in comparison with control group (p < 0.05). Consistently, the content of C3a and C5a in liver increased markedly in mice with aGVHD (p < 0.01). For animals ongoing aGVHD, complement component C3 depositions were observed in hepatic portal areas, around which massive inflammatory cell infiltration was also observed. It is concluded that in aGVHD animals, excessive complement activation occurs, and the activated complement components participate in pathological process of the aGVHD.


Assuntos
Ativação do Complemento , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Animais , Transplante de Medula Óssea , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
11.
Transpl Immunol ; 24(1): 17-25, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20850528

RESUMO

Teff/Treg imbalance orchestrated the onset and the progression of the lupus nephritis in a DBA/2→B6D2F1 murine model with cGVHD. In this paper, we first used 145-2C11 Ab to treat these human SLE-like diseased animals. The results showed that short-term low-dose anti-CD3 antibody treatment induced a significant remission of established proteinuria, production of autoantibodies, immune complex deposition and renal parenchyma lesions in lupus nephritic mice. Of note, we found a robust up-regulation of Foxp3 mRNA expression in the target tissue: kidney from mice with anti-CD3 antibody treatment compared to those with control IgG treatment. Likewise, an increased renal mRNA abundance for IL-10 was also observed in anti-CD3 antibody treated mice. In contrast, genes associated with inflammation and fibrosis as well as cytokines related to effector T cell responses were down-regulated by anti-CD3 mAb treatment. These findings suggested that short-term low-dose anti-CD3 antibody treatment might induced an IL-10-secreting Foxp3(+) regulatory T cells in this cGVHD target tissue: kidney, that suppressed the activation of effector T cells (Th1, Th2 and Th17), thus ameliorating the severity of the lupus nephritis in mice.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Doença Enxerto-Hospedeiro/tratamento farmacológico , Rim/metabolismo , Nefrite Lúpica/tratamento farmacológico , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Complexo CD3/imunologia , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/complicações , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/fisiopatologia , Humanos , Terapia de Imunossupressão , Interleucina-10/genética , Interleucina-10/metabolismo , Rim/efeitos dos fármacos , Rim/imunologia , Rim/patologia , Nefrite Lúpica/etiologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/fisiopatologia , Camundongos , Camundongos Endogâmicos DBA , Proteinúria , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Regulação para Cima
12.
Microbes Infect ; 12(12-13): 948-55, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20601078

RESUMO

A novel human antibody AR16, targeting the G5 linear epitope of rabies virus glycoprotein (RVG) was shown to have promising antivirus potency. Using AR16, the minimal binding region within G5 was identified as HDFR (residues 261-264), with key residues HDF (residues 261-263) identified by alanine replacement scanning. The key HDF was highly conserved within phylogroup I Lyssaviruses but not those in phylogroup II. Using computer-aided docking and interaction models, not only the key residues (Asp30, Asp31, Tyr32, Trp53, Asn54, Glu99, Ile101, and Trp166) of AR16 that participated in the interaction with G5 were identified, the van der Waals forces that mediated the epitope-antibody interaction were also revealed. Seven out of eight presumed key residues (Asp30, Asp31, Tyr32, Trp53, Asn54, Glu99, and Ile101) were located at the variable regions of AR16 heavy chains. A novel mAb cocktail containing AR16 and CR57, has the potential to recognize non-overlapping, non-competing epitopes, and neutralize a broad range of rabies virus.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Vírus da Raiva/imunologia , Substituição de Aminoácidos , Sítios de Ligação , Sequência Conservada , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica
13.
Int J Biomed Sci ; 5(4): 336-44, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23675156

RESUMO

Using computer-guided homology modeling method, the 3-D structure of the Fv fragment of a functional anti-IgE antibody (MAE11) was constructed and the spatial structure of E24-MAE11 complex was modeled based on the crystal structure of IgE-Fc (abbr. E24) and molecular docking method. Then the identified epitope of IgE was determined theoretically, which showed the key role of IgE-Cɛ3 in interacting with both FcɛRIα and MAE11. By normal protocols, we immunized mice with purified protein E34 and screened six anti-E34 monoclonal antibodies. Purified antibodies could identify E34 by Western blot; furthermore, all of them could bind IgE by ELISA, in which QME5 seemed to be the best. Flow cytometry analysis displayed that only QME5 could bind membrane IgE and it could compete with membrane FcɛRIα to bind soluble IgE. Meanwhile, QME5 couldn't bind FcɛRIα-attached IgE, which suggested no hypersensitivity in triggering the target cells (mast cells or basophils) by crosslinking or inducing the release of a variety of chemical mediators.

14.
J Biomol Screen ; 12(6): 800-8, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17644770

RESUMO

In this study, the authors apply a computer-based strategy to screen thousands of small-molecule, nonpeptidic organic compounds in the Available Chemicals Directory database and to select a series of potential candidates as ligands of the proposed CD4 D1 surface pocket. Then, several cell-based models are used to determine the actual biological functions of these compounds. A small molecule designated A5 (N-((pyridine-4-yl)methylene)thiophene-2-carbohydrazide) was obtained by a virtual screening followed by 3 cell-based functional assays. The results show that A5 could specifically block the CD4-major histocompatibility complex II binding in a rosetting assay, inhibit the mixed lymphocyte reaction-induced T-cell proliferation in a concentration-dependent manner, and reduce the PMA plus ionomycin-stimulated interleukin-2 secretion from peripheral blood mononuclear cells.


Assuntos
Antígenos CD4/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Desenho Assistido por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Piridinas/farmacologia , Tiofenos/farmacologia , Algoritmos , Linfócitos T CD4-Positivos/fisiologia , Células Cultivadas , Humanos , Interleucina-2/metabolismo , Teste de Cultura Mista de Linfócitos , Modelos Biológicos , Modelos Moleculares , Formação de Roseta , Tienopiridinas
15.
Mol Immunol ; 44(5): 784-95, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16787663

RESUMO

The interaction between CD4 and major histocompatibility complex (MHC) class II proteins is critical for the activation of CD4+ T cells, which are involved in transplantation reactions and a number of autoimmune diseases. It is known that the CD4 N-terminal immunoglobulin variable region-like domain (D1) is directed toward and reaching into the two membrane-proximal domains of the MHC class II molecule. Thus, compounds targeted to D1 would be expected to function as the inhibitors of the interaction of CD4 and class II MHC molecules. In this study, we used a computer-based design method to screen thousands of non-peptidic compounds in a molecular database and identified a group of compounds as potential ligands of CD4 D1. These small organic compounds were then synthesized and tested by actual biological assays. One of them, named J2, which possessed favorable activity, was obtained. Experimental data showed that J2 could specifically block stable CD4-MHC class II binding and elicit significant inhibition of immune responses in vitro and in vivo. All the results demonstrated the therapeutic potential of this compound as a novel immunosuppressive agent.


Assuntos
Acrilamidas/farmacologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Região Variável de Imunoglobulina/imunologia , Imunossupressores/farmacologia , Indóis/farmacologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Epitopos de Linfócito T/imunologia , Feminino , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Interleucina-2/imunologia , Ligantes , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Pele/imunologia
16.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 22(3): 363-7, 2006 May.
Artigo em Chinês | MEDLINE | ID: mdl-16643801

RESUMO

AIM: To construct the eukaryotic expression vector of chimeric anti-human CD20 monoclonal antibody (mAb) and realize its expression. METHODS: The light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR and were cloned to T vector and sequenced. Proteins of mAb 1-28 were separated by reducing SDS-PAGE. Light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. Software Biolynx and pepeseq were used to evaluate the score of probability. Correctness of the light- and heavy-chain DNA sequences was verified by their protein sequences. Genes of V(H) and V(L) were amplified from T vector and cloned into chimeric antibody expression vector (pCMV-V(H) and pCMV-V(L)), generating the expression vectors of chimeric anti-human CD20 mAb (C1-28) including light chain expression vector C1-28L and heavy chain expression vector C1-28H. The two plasmids were co-transfected into 293T cells with Lipofectamine 2000. RT-PCR was used to detect the transcription at mRNA level. C1-28 expression was detected by Sandwich ELISA and Western blot methods. RESULTS: mAb 1-28's genes were successfully cloned and verified by peptide mass fingerprinting. Eukaryotic expression vectors of anti-human CD20 mAb were constructed and expressed in 293T cells with the expression amount reaching 257 mg/L and the molecular weight consistent with that of human IgG. CONCLUSION: These experiments lay solid foundation for further study on the role of chimeric CD20 antibody in the treatment of non-Hodgkin's lymphoma.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Sistemas de Liberação de Medicamentos , Hibridomas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Antígenos CD20/genética , Células Cultivadas , Quimera , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Humanos , Hibridomas/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Linfoma não Hodgkin/terapia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Rituximab , Transfecção
17.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 22(2): 171-3, 2006 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-16507251

RESUMO

AIM: To clone and express soluble B lymphocyte stimulator (sBLyS). METHODS: Total RNA was isolated from peripheral blood mononuclear cells, and used to synthesize cDNA by reverse transcription. sBLyS cDNA was amplified by PCR with specific primers and inserted into a prokaryotic expression vector pET-30a. Recombinant plasmid was transformed into E.coli strain BL21(DE3). sBLyS was expressed in E.coli, purified in vitro, and analyzed with peptide mass fingerprinting and Daudi cell proliferation assay. RESULTS: sBLyS cDNA was cloned. Peptide mass fingerprinting of purified BLyS matched with that of BLyS proteins. Purified sBLyS could stimulate Daudi cell proliferation in vitro. CONCLUSION: sBLyS with biological activity was successfully expressed and purified.


Assuntos
Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Expressão Gênica , Leucócitos Mononucleares/química , Animais , Clonagem Molecular , DNA Complementar/análise , Amplificação de Genes , Vetores Genéticos/genética , Humanos , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da Polimerase
18.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 22(1): 64-6, 70, 2006 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-16388748

RESUMO

AIM: To gain recombinant protein Cepsilon3-Cepsilon4 of IgE Fc (E34). METHODS: We cloned the gene coding human IgE Cepsilon3-Cepsilon4 (E34) and constructed an expression vector pET28a(+)-E34. The target protein was expressed as inclusion body in E.coli BL-21. Following renaturation and purification through a CM sephorose FF column, the soluble protein was acquired, and its binding ability to murine anti-hIgE mAb was identified by Western blot and ELISA. RESULTS: The cloned E34 gene was sequenced and proved by SDS-PAGE to be the same as reported sequence. SDS-PAGE analysis showed the relative molecular mass of E34 protein obtained was correct as predicted. Western blot and ELISA data revealed that it owned the epitope of binding to murine anti-hIgE mAb. CONCLUSION: The expression vector pET28a(+)-E34 has been successfully constructed and the target protein E34 recognized specifically by murine anti-hIgE mAb is obtained.


Assuntos
Regulação da Expressão Gênica , Imunoglobulina E/isolamento & purificação , Imunoglobulina E/metabolismo , Anticorpos Monoclonais/metabolismo , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/genética , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Hybridoma (Larchmt) ; 24(5): 263-6, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16225427

RESUMO

One strain of neutralizing monoclonal antibody (MAb) against cell-binding polypeptide of ricin, named 3E1, was generated efficiently. The antibody recognized the linearity epitope of RTB located in a toxin structure domain characterized by Western blotting. The safe period of mice for intraperitoneal injection of 100 microg of antibody was 20 min after intraperitoneal injection of 2 microg of Ricin (10 times LD50). The neutralizing MAb we obtained could be developed into an immunotherapeutic agent to counteract the use of ricin as a terrorist or biological warfare weapon. It might be useful, as well, for antibody-based prophylaxis.


Assuntos
Anticorpos Monoclonais/imunologia , Peptídeos/imunologia , Ricina/imunologia , Animais , Linhagem Celular , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização/métodos , Peptídeos/antagonistas & inibidores , Peptídeos/toxicidade , Ricina/antagonistas & inibidores , Ricina/toxicidade , Testes de Toxicidade Aguda
20.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 21(4): 466-9, 2005 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-15989793

RESUMO

AIM: To seek a good method for simultaneous cloning V(L), V(H) and signal peptide genes of anti-CD20 monoclonal antibody (mAb) from hybridoma cells 1-28. METHODS: Total RNA was prepared from hybridoma cells 1-28 by Trizol. Two methods were used to clone the target genes. One was traditional rapid amplification of 5' cDNA end (T-5' RACE), and the other was RNA ligase-mediated rapid amplification of 5' cDNA end (RLM-RACE). The PCR products were then cloned into pGEM-T Easy vector, sequenced and analyzed with Blast method using Kabat and GenBank Databases. RESULTS: The target genes of L chain and H chain were both successfully cloned by RLM-RACE, while only V(L) gene and its signal peptide genes were obtained by T-5' RACE. CONCLUSION: RLM-RACE is a good method for cloning antibody's V gene and signal peptide genes.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase/métodos , Sinais Direcionadores de Proteínas/genética , RNA Ligase (ATP)/metabolismo , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Sequência de Bases , Clonagem Molecular , Eletroforese em Gel de Ágar , Humanos , Região Variável de Imunoglobulina/química , Dados de Sequência Molecular , RNA/análise , RNA/genética , RNA/metabolismo
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