RESUMEN
Generation of protein immunogens is often a rate-limiting step in the production of monoclonal antibodies (Mabs). Expressing domains of proteins as fusions to the baculovirus surface glycoprotein gp64 displays foreign proteins on the surface of the virion. Antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the gp64 nucleotide sequence. This method allows immunization with whole virus, eliminating the need for purification of target antigens. Affinity-matured Mabs to the human nuclear receptors LXRbeta and FXR have been produced using baculovirus particles displaying gp64/nuclear receptor fusion proteins as the immunizing agent. Immunizations were performed directly with pelleted virus using the Repetitive Immunization Multiple Sites (RIMMS) immunization strategy for rapid Mab production. All Mabs were identified using insect cells infected with the immunizing virus. Characterization of these antibodies shows them to be class-switched and specific for LXRbeta or FXR. Additionally, high affinity antibodies that recognize gp64 and neutralize baculovirus infection of insect cells were isolated. Use of the recombinant baculovirus gp64 display system makes possible the production of Mabs once a partial DNA sequence is known. This allows the generation of antibodies prior to the isolation of purified protein, in turn providing antibodies to facilitate purification, characterization and immunolocalization of proteins.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Proteínas de Unión al ADN/inmunología , Vectores Genéticos , Nucleopoliedrovirus , Receptores Citoplasmáticos y Nucleares/inmunología , Factores de Transcripción/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Proteínas de Unión al ADN/genética , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Immunoblotting/métodos , Inmunohistoquímica/métodos , Receptores X del Hígado , Ratones , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/inmunología , Nucleopoliedrovirus/patogenicidad , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de Transcripción/genética , Proteínas Virales de Fusión/genéticaRESUMEN
A cDNA clone encoding bovine muscarinic acetylcholine m3 receptor has been isolated from a bovine brain library. Sequencing of this clone has revealed a single open reading frame encoding a protein of 590 amino acids. Comparison with known muscarinic m3 receptor sequences from other species shows a high degree of conservation (92-98% homology) in the protein sequence. COS cells transfected with this cDNA expressed a single high-affinity (Kd = 43.9 pM) binding site for muscarinic receptor ligand N-[3H]methylscopolamine.
Asunto(s)
ADN Complementario/genética , Receptores Muscarínicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Clonación Molecular , ADN Complementario/química , Datos de Secuencia Molecular , Receptores Muscarínicos/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16)] and an intercalator [4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA)] was studied as a function of proliferation in Chinese hamster ovary (CHO), HeLa, and mouse leukemia L1210 cell lines. Log-phase CHO cells exhibited dose-dependent drug-induced DNA breaks, while plateau cells were found to be resistant to the effects of VP-16 and m-AMSA. Neither decreased viability nor altered drug uptake accounted for the drug resistance of these confluent cells. In contrast to CHO cells, plateau-phase HeLa and L1210 cells remained sensitive to VP-16 and m-AMSA. Recovery of drug sensitivity by plateau-phase CHO cells was found to reach a maximum approximately 18 h after these cells regained exponential growth and was independent of DNA synthesis. DNA strand break frequency correlated with cytotoxicity in CHO cells; log cells demonstrated an inverse log linear relationship between drug dose (or DNA damage) and colony survival, whereas plateau-derived colony survival was virtually unaffected by increasing drug dose. Topoisomerase II activity, whether determined by decatenation of kinetoplast DNA, by cleavage of pBR322 DNA, or by precipitation of the DNA-topoisomerase II complex, was uniformly severalfold greater in log-phase CHO cells compared to plateau-phase cells.
