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. 2003 Jan 15;85(2):413–420. doi: 10.1016/0378-1119(89)90434-4

A general method for the induction and screening of antisera for cDNA-encoded polypeptides: antibodies specific for a coronavirus putative polymerase-encoding gene

Philip W Zoltick a, Julian L Leibowitz b, James R DeVries b, George M Weinstock c, Susan R Weiss a,∗
PMCID: PMC7127337  PMID: 2560756

Abstract

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 β-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.

Keywords: Recombinant DNA, open reading frame vector, nonstructural viral proteins, cell-free protein synthesis, mouse hepatitis virus

Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); βGal, β-galactosidase; B/V, bacterial/viral (fusion protein); CAT, Cm acetyl transferase; cat, gene encoding CAT; cDNA, DNA complementary to RNA; Cm, chloramphenicol; IPTG, isopropyl-β-d-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; MHV, mouse hepatitis virus; moi, multiplicity of infection; NP40, Nonidet P40; nt, nucleotide(s); ONPG, o-nitrophenyl-d-galactopyranoside; ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PBS, 0.9% NaCl/10mM Na · phosphate pH 7.4; PMSF, phenylmethylsulfonyl fluoride; 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; SDS, sodium dodecyl sulfate; TS, 10 mM Tris pH 7.4/10 mM NaCl/1.5 mM MgCl2; TS/P, TS with 20 μg PMSF/ml; wt, wild type; XGal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside

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