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发现的霍乱弧菌共同结构域hlyA进行了定点诱变。将该结构域的一个氨基酸取代为另一个氨基酸，合成了寡核苷酸引物。这些突变被设计为替代另一种氨基酸，其中(A)亲水性和(B)电荷被极大地改变，(C)密码子的第三个核苷酸被改变，从而产生氨基酸取代。利用这些寡核苷酸引物进行定点突变，并通过DNA测序证实突变，得到10个突变体。突变体前体在大肠杆菌中过量产生。提取并部分纯化了突变体溶细胞素。测定细胞溶素抗原的数量和蛋白含量。突变体溶细胞素的比活性为天然溶细胞素的1/10 ~ 1/100。这表明霍乱弧菌和创伤弧菌中存在的共同结构域在溶细胞素的溶细胞活性中起重要作用。

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.