Assuntos
Sequência de Bases , DNA Recombinante , Reação em Cadeia da Polimerase/métodos , Animais , Linhagem Celular , Clonagem Molecular/métodos , Enzimas de Restrição do DNA , DNA Recombinante/isolamento & purificação , DNA Polimerase Dirigida por DNA , Eletroforese em Gel de Ágar , Vetores Genéticos , Indicadores e Reagentes , Mariposas , Oligodesoxirribonucleotídeos , Plasmídeos , Taq Polimerase , TransfecçãoRESUMO
To study the interaction of human factor VIII (FVIII) with its various ligands, select regions of cDNA encoding FVIII light chain were cloned into the plasmid expression vector pET3B to overproduce FVIII protein fragments in the bacterium Escherichia coli. Partially purified FVIII protein fragments were used to produce monoclonal antibodies. One monoclonal antibody, 60-B, bound both an FVIII protein fragment (amino acid residues 1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This antibody prevented FVIII-vWF binding and acted as an inhibitor in both the activated partial thromboplastin time (APTT) assay and a chromogenic substrate assay that measured factor Xa generation. The ability of the antibody to inhibit FVIII activity was diminished in a dose-dependent fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino acid residues 1674 through 1684. The 60-B antibody did not react with a peptide in which the aspartic acid residue at 1681 (underlined) was changed to a glycine, which is the amino acid present at this position in porcine FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human FVIII showed that the 60-B antibody prevented thrombin cleavage at light chain residue 1689. The coagulant inhibitory activity of the 60-B antibody may be due, in part, to the prevention of thrombin activation of FVIII light chain.
Assuntos
Anticorpos Monoclonais/farmacologia , Fator VIII/imunologia , Trombina/metabolismo , Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Western Blotting , Clonagem Molecular , Escherichia coli/genética , Fator VIII/genética , Fator VIII/metabolismo , Polarização de Fluorescência , Humanos , Dados de Sequência Molecular , Tempo de Tromboplastina Parcial , Fragmentos de Peptídeos/imunologia , Plasmídeos , TripsinaRESUMO
We have developed a polymerase chain reaction (PCR) based procedure for rapidly analyzing recombinant vectors in whole bacterial cells. No purification, restriction mapping or sequencing of vectors is required and the results are available within 6 hours. Whole cells are added to a PCR mix that is designed to amplify DNA only if the correct insert is present in the required orientation. The presence of an appropriately sized band on an agarose gel is indicative of a correct clone.
Assuntos
Escherichia coli/genética , Vetores Genéticos , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Biotecnologia , DNA/genética , DNA Bacteriano/genética , Fator VIII/genética , Humanos , Recombinação Genética , Transformação GenéticaRESUMO
We have used a microcomputer program to test eukaryotic mRNA 5'-leader sequences for complementarity to the 3'-terminus of 18S rRNA. No mismatched bases, bulge loops, or viral mRNA's were utilized. At least one-fourth of the more than 200 mRNA's studied were found to have two distinct regions of complementarity which resulted in an ability to bind to two separate rRNA regions (GAAGG and UUUGG). The analysis of 60 mRNAs with these dual sites resulted in a consensus structure that was a mean distance of 11.75 bases 5' from the initiator AUG and had an average predicted interstrand binding strength of delta G = -13.40 kcal. These characteristics compare favorably to those observed for the prokaryotic 16S rRNA-mRNA Shine and Dalgarno bond.
