- 期刊
Cloning, Sequencing and Expression of a GroEL-like Protein Gene of Vibrio vulnificus
創傷弧菌類GroEL蛋白質基因之選殖、定序和表現
摘要
創傷弧菌造成人類的傷口感染，或者透過食用污染的海產而導致敗血症，死亡率高。本研究選殖了該菌的類GroEL蛋白質基因，核酸定序與特性分析。將V. vulnificus ATCC27562核酸增幅得到一個1.6kb片段，轉殖至pGEM-T載體，完成核酸定序。該片段含有一個完整的l,644bp open reading frame，會產生一個57.7kDa的類GroEL蛋白質，共547個胺基酸。推演所得的胺基酸序列很近似其他弧菌、大腸桿菌和沙門氏菌的GroEL蛋白質，不過在第269至282個胺基酸片段顯著地比其他同類蛋白質較為斥水性。將這個基因轉殖至表現載體pQE-30，生成具有His標記的類GroEL蛋白質，使用抗GroEL和抗His抗體檢測到該轉殖蛋白質大量生成，而且是以不溶解狀態存在。本研究成果有助於瞭解創傷弧菌對環境壓力之反應。
並列摘要
Vibrio vulnificus causes wound infection and septicemia in humans with high mortality via the consumption of contaminated seafood. This study cloned, sequenced and analyzed the general properties of the GroEL-like protein gene of this pathogen. A 1.6 kb amplified DNA fragment from V. vulnificus ATCC27562 was cloned into pGEM-T Vector and nucleotide sequenced. The open reading frame consisted of 1,644bp, and encoded for a 57.7kDa GroEL-like protein of 547 amino acids. The amino acid sequence of the V. vulnificus GroEL-like protein showed high identity (82-92%) with other GroEL proteins, especially those of vibrios, E. coli and S. enterica Typhi. The amino acid residues 269 to 282 of the V. vulnificus GroEL-like protein were significantly more hydrophobic than others, with two basic lysine residues replaced by hydrophobic isoleucine residues. The groEL-like gene of V. vulnificus was cloned into the pQE-30 expression vector and expression of a His-tagged GroEL-like protein was rapidly induced by isopropyl-β-D- thiogalactopyranoside and confirmed by immunoblotting with anti-GroEL and Penta-His antibodies. The over-produced GroEL-like protein of V. vulnificus was present largely in an insoluble form. Results of this report may facilitate the study of responses of V. vulnificus to environmental stresses.