RESUMO
In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Anticorpos de Cadeia Única/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaAssuntos
Fator V/genética , Epidemiologia Molecular , Protrombina/genética , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Prevalência , Tunísia/epidemiologiaRESUMO
Human neutrophil antigens play an important role in provoking immune neutropenia and transfusion-reactions. The aim of this study was to determine granulocyte-specific antigens on the neutrophil Fc gamma receptor IIIb (Fc gamma RIIIb, CD16b), namely, the HNA-1a(NA1) and HNA-1b(NA2) antigens and their gene frequencies in Tunisian blood donors and Berbers. One hundred and ninety-nine unrelated healthy Tunisian blood donors and Berbers were typed for HNA-1a and HNA-1b(NA1 and NA2), using polymerase chain reaction with sequence-specific primers (PCR-SSP). In 24 granulocyte samples, the HNA-1a and HNA-1b phenotypes was additionally determined by the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results. A subsequent analysis of the genotyping study showed that, the HNA-1a and HNA-1b gene frequencies observed, were 0.342 and 0.658 for Berbers, and 0.311 and 0.668 for blood donors, respectively. In the genotyping study conducted, it was determined that the HNA-1a and HNA-1b gene frequencies observed in Tunisian blood donors and Berbers are similar to those previously reported in other white populations.