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

对创伤弧菌溶细胞素中霍乱弧菌常见结构域的hlyA进行了定点突变。目的：1.合成寡核苷酸引物，将该结构域的一种氨基酸替换为另一种氨基酸，使其发生突变，使其(A)亲水性和(B)电荷发生较大变化，(C)密码子的第三个核苷酸发生氨基酸替换。利用这些寡核苷酸引物进行定点突变，DNA测序证实突变，获得10个突变体。突变前体在E.Coli中过量生产。对突变型细胞溶血素进行提取和部分纯化。测定细胞溶血素抗原的含量和蛋白质含量。突变株细胞溶血素的比活力为天然细胞溶血素的1/10~1/100。这一结果表明霍乱弧菌和创伤弧菌的共同结构域在溶细胞素的细胞溶解活性中起着重要作用(S)。

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.