Site-Directed Mutagenesis of HlyA of Vibrio Cholerae at Common Domain Found in Vibroio Vulnificus Cytolysin.

点击翻译按钮获取中文摘要

Site-directed mutagenesis of hlyA of Vibrio cholerae at common domain found in Vibrio vulnificus cytolysin was carried out.1. Oligonucleotide primers were synthesized as one of the amino acids of the domain in substituted to another amino acid.The mutations were designed to substitute to another amino acid of which(A) hydropathy and(B) charge was greatly changed and(C) the third nucleotide of the codons was changedwhere it gives amino acid substitution.2. Site-directed mutations were carried out using these oligonucleotide primers and the mutation was confirmed by DNA sequencing and ten mutants were obtained.3. The mutant precursors were over-produced in E. Coli. The mutant cytolysins were extracted and partially purified. The quantities of the cytolysin antigens and the protein contents were determined.4. The specific activities of the mutant cytolysins were 1/10 - 1/100 of the native cytolysin. This result suggest that the common domain found in V. Cholerae and V. Vulnificus play an important role(s) in cytolytic activities of the cytolysins.