RESUMO
We describe the chimerization of a monoclonal antibody directed against the c-erbB-2 protein using a novel PCR method for cloning immunoglobulin variable region genes. We also describe the characterization of the chimera and show its potential use for treating cancers which overexpress the c-erbB-2 protein. The genomic DNA fragments of heavy and light chain variable genes were cloned by PCR using uniquely designed primers which allowed for isolation of genes containing functional promoters, signal and coding sequences. The chimeric genes were then constructed by linking variable regions of murine genes to human constant gamma 1 and kappa genes. Expression of the chimeric immunoglobulin genes resulted in production of properly assembled chimeric antibody with improved biological properties.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Neoplasias Experimentais/terapia , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Clonagem Molecular , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The herpes simplex virus type 2 (HSV-2) genome codes for an envelope protein, glycoprotein G (gG), which contains predominantly type 2-specific epitopes. A portion of this gG gene has been expressed as a fusion protein in Escherichia coli. Expression was regulated by a lambda phage pL promoter. The 60,000-molecular-weight recombinant protein was purified by ion-exchange chromatography. Amino acid sequence analysis confirmed the N terminus of the purified protein. Mice immunized with recombinant gG developed antibodies reactive with native HSV-2 protein, but not with HSV-1 protein, in an indirect immunofluorescence assay. The serological activity of this purified recombinant gG protein was evaluated by immunoblot assay. This protein was reactive with an HSV-2 gG monoclonal antibody. It was also reactive with HSV-2 rabbit antiserum but not with HSV-1 rabbit antiserum. Of 15 patient serum samples known to have antibody to HSV-2, 14 were reactive with this recombinant type 2-specific gG protein, and none of 15 HSV antibody-negative patient serum samples showed reactivity. In agreement with the expected prevalence of HSV-2 infection, 27.6% of 134 serum samples from random normal individuals had antibodies reactive with recombinant gG. This recombinant gG protein may be of value in detecting HSV-2-specific antibody responses in patients infected with HSV-2.
Assuntos
Simplexvirus/genética , Proteínas do Envelope Viral/genética , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/biossíntese , Bacteriófago lambda/genética , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Genes Virais , Herpes Simples/genética , Herpes Simples/imunologia , Humanos , Peso Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Simplexvirus/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
Three proteins (env, gag, and tax) encoded by the human T-cell leukemia virus type I (HTLV-I) genome were cloned and expressed in Escherichia coli. The env protein contained a substantial part of the gp46 domain and a majority of the p21e domain. The gag protein contained all of p24 and portions of p19 and p15. In addition to these two structural proteins, a full-length tax (p40X) construct was obtained. All three recombinant proteins were purified to near homogeneity. When used in an immunoblot assay, the three recombinant proteins detected antibodies in more HTLV-I antibody-positive patient sera than did the corresponding native proteins. Antibodies to at least two of these three different gene products were detected in 98.4% of adult T-cell leukemia patients, 100% of HTLV-I-associated myelopathy patients, 97.4% of asymptomatic carriers, and 94% of uncharacterized HTLV-I-positive patients. Antibody to recombinant tax was found in 4.9% of adult T-cell leukemia patients, whereas antibody to recombinant env could not be detected. These recombinant proteins from three different gene products may be useful in detecting or confirming the presence of antibodies to HTLV-I.