A general method for the induction and screening of antisera for cDNA-encoded polypeptides: antibodies specific for a coronavirus putative polymerase-encoding gene
| Autor: | Philip W. Zoltick, George M. Weinstock, Susan R. Weiss, James R. Devries, Julian L. Leibowitz |
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| Rok vydání: | 1989 |
| Předmět: |
viruses
medicine.disease_cause Cm chloramphenicol Mice bp base pair(s) Gene expression Cloning Molecular aa amino acid(s) Polymerase Coronavirus ONPG o-nitrophenyl-d-galactopyranoside Expression vector cDNA DNA complementary to RNA biology TS 10 mM Tris pH 7.4/10 mM NaCl/1.5 mM MgCl2 virus diseases DNA-Directed RNA Polymerases General Medicine open reading frame vector cell-free protein synthesis cat gene encoding CAT wt wild type Plasmids SDS sodium dodecyl sulfate moi multiplicity of infection Viral protein Recombinant Fusion Proteins XGal 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside Genetic Vectors Molecular Sequence Data kb kilobase(s) or 1000 bp PAGE polyacrylamide-gel electrophoresis CAT Cm acetyl transferase PBS 0.9% NaCl/10mM Na · phosphate pH 7.4 IPTG isopropyl-β-d-thiogalactopyranoside Antibodies Article Cell Line TS/P TS with 20 μg PMSF/ml Gene product RIPA buffer 0.1 % SDS/1 % NP40/400 mM NaCl/25 μg PMSF per ml/20 μg aprotinin per ml/10 mM Na · phosphate pH 7.4 ORF open reading frame Complementary DNA Genetics medicine Animals Ap ampicillin MHV mouse hepatitis virus Murine hepatitis virus Recombinant DNA mouse hepatitis virus Base Sequence NP40 Nonidet P40 βGal β-galactosidase Fusion protein Virology Molecular biology Rec A Recombinases nonstructural viral proteins PMSF phenylmethylsulfonyl fluoride biology.protein nt nucleotide(s) B/V bacterial/viral (fusion protein) |
| Zdroj: | Gene |
| ISSN: | 0378-1119 |
| DOI: | 10.1016/0378-1119(89)90434-4 |
| Popis: | A prokaryotic vector, pGE374, containing the recA and lacZ genes, out-of-frame, was used for the expression of cDNA derived from the putative polymerase-encoding gene of the coronavirus mouse hepatitis virus strain A59 (MHV-A59). The pGE374/viral recombinant vector generates a tripartite bacterial/viral protein composed of a segment of the RecA protein at the N terminus, the coronaviral sequences in the middle, and an enzymatically active beta-galactosidase at the C terminus. Rabbits immunized with such recombinant proteins generated antibodies to the MHV-A59 portion of the tripartite protein. Because the MHV-A59 polymerase proteins have been difficult to identify during infection, we used a novel method to demonstrate the viral specificity of the antiserum. The viral cDNA was excised from the expression vector, and transferred to a pGem vector, downstream from and in-frame with a portion of the cat gene. This construct contained a bacteriophage RNA polymerase promoter that enabled the cell-free synthesis of a fusion protein that was used to verify that antibodies were generated to the expressed viral DNA. This strategy was shown to successfully result in the specific generation of antibodies to the encoded information of the viral cDNA. Furthermore, this method has general applicability in the generation and characterization of antibodies directed against proteins encoded in cDNAs. |
| Databáze: | OpenAIRE |
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