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1.
Artigo em Chinês | WPRIM (Pacífico Ocidental) | ID: wpr-299277

RESUMO

<p><b>OBJECTIVE</b>To establish an ex vivo model of myocardial ischemia reperfusion in tree shrews.</p><p><b>METHODS</b>The Langendorff ex vivo heart perfusion system was used to establish the myocardial ischemia reperfusion model in tree shrews with different irrigation and reperfusion time settings. Alanine aminotransferase (ALT), aspartate transaminase (AST) and lactic dehydrogenase (LDH) levels were measured by enzyme-labeled immunosorbent assay, creatine kinase MB (CK-MB) was detected using immunosuppression method, and malondialdehyde was measured with thiobarbital staining method; the infarct size was measured using 2, 3, 5-triphenyltrazoliumchloride (TTC) method.</p><p><b>RESULTS</b>Ischemia for 30 min and reperfusion for 30 and 60 min caused more significant increase in CK-MB and LDH levels in the perfusion fluid and also in the levels of ALT, CK-MB and AST in the myocardial tissue compared with other experimental settings (P<0.05), but these parameters were comparable between the former two settings (P>0.05). The mean heart rate in 30-min ischemia with 60-min reperfusion group was obviously lower than that in continuous reperfusion group, 15-min ischemia with 30-min reperfusion group and 30-min ischemia with 30-min reperfusion group (P<0.05), and the heart rate was similar between the latter 3 groups (P>0.05). ECG analysis showed that the mean heart rate in 30-min ischemia with 30-min reperfusion group was closer to the physiological heart rate of tree shrews.</p><p><b>CONCLUSION</b>We successfully established an ex vivo myocardial ischemia reperfusion model using tree shrews, and ischemia for 30 min followed by reperfusion for 30 min is the optimal experimental setting.</p>

2.
Acta Biochim Biophys Sin (Shanghai) ; 37(6): 415-20, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15944757

RESUMO

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS. After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.


Assuntos
Anticorpos/imunologia , Linfócitos B/imunologia , Região Variável de Imunoglobulina/imunologia , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Fator Ativador de Células B , Antígeno de Maturação de Linfócitos B , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Biblioteca de Peptídeos , Fator de Necrose Tumoral alfa/antagonistas & inibidores
3.
Acta Biochim Biophys Sin (Shanghai) ; 36(8): 541-7, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15295646

RESUMO

Three single chain antibodies (scFv) against the proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated by phage display from an scFv antibody library. Bio-panning was carried out against immobilized purified envelope (E) and nucleocapsid (N) proteins of SARS-CoV. Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA. Two of them, B10 and C20, could recognize non-overlapping epitopes of the E protein according to the two-site binding test result. Clone A17 could recognize N protein. The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ. The affinity (equilibrium dissociation constant, K(d)) of SARS-CoV E protein was 5.7 x 10(-8) M for B10 and 8.9 x 10(-8) M for C20. The affinity of A17 for N protein was 2.1 x 10(-6) M. All three scFv antibodies were purified with affinity chromatography and determined by Western blot.


Assuntos
Anticorpos Antivirais , Produtos do Gene env/imunologia , Proteínas do Nucleocapsídeo/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Proteínas do Nucleocapsídeo de Coronavírus , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Técnicas In Vitro , Cinética , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos
4.
Artigo em Chinês | MEDLINE | ID: mdl-14673506

RESUMO

The cDNA of human B lymphocyte stimulator C-terminal peptide (C-BLyS) was amplified by nested PCR from cDNA library of human fetal brain. The expression plasmid pT7450-C-BLyS was constructed and transformed into E. coli BL21 CodonPlus (DE3) RIL which can recognize many rare codons. The C-BLyS protein was expressed as inclusion body in E. coli BL21 CodonPlus (DE3) RIL and the inclusion body of C-BLyS was denatured and then refolded by dialysis and purified by ion exchange chromatography. The refolded and purified C-BLyS can specifically bind with BCMA-Fc, a fusion protein of BLyS receptor and human IgG1 Fc. Furthermore, C-BLyS is proved to be an effective stimulator in mouse splenocytes proliferation in vitro and effective immunostimulant in vivo.


Assuntos
Proteínas de Membrana/imunologia , Proteínas Recombinantes/isolamento & purificação , Fator de Necrose Tumoral alfa/imunologia , Animais , Fator Ativador de Células B , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Cromatografia por Troca Iônica , Relação Dose-Resposta a Droga , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Camundongos , Muramidase/imunologia , Muramidase/metabolismo , Dobramento de Proteína , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia
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