Use of degenerate primers and heat-soaked polymerase chain reaction (PCR) to clone a serine protease antigen from Dermatophilus congolensis.
Serine proteases are thought to be involved in the initial attack on sheep skin by Dermatophilus congolensis and are obvious antigens for inclusion in a vaccine to prevent lumpy wool disease (dermatophilosis). Degenerate primers were designed after alignment of seven bacterial serine proteases. Inosine was incorporated into the primers at positions of three- and four-base redundancy, and this reduced the complexity of the primer mixtures from several thousand to sixteen different sequences for each primer. The primers were validated by production and sequencing of amplicons from serine protease genes in Bacillus subtilis and Serratia marcescens. The primers were used with heat-soaked polymerase chain reaction (PCR) to produce amplicons from two D. congolensis strains, AG and MB. In the amplicon codons for arginine, rather than the expected serine, were found where inosine was used for both the first and third positions for a codon in the primer. A search with the deduced amino acid sequences of the amplicons showed significant similarity to a keratinase and other serine proteases from various organisms. Similarity was most apparent around the active site residues and other essential secondary structural elements.
Mine
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State Agricultural Biotechnology Centre, Murdoch University, Perth, Australia. ommine@temo.bca.bw
Carnegie
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PR
D000192:Actinomycetales / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence / Q000235:genetics; D000942:Antigens, Bacterial / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000302:isolation & purification; D005784:Gene Amplification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000235:genetics* / Q000276:immunology; D012706:Serratia marcescens / Q000235:genetics
D000942:Antigens, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D012697:Serine Endopeptidases