透明颤菌血红蛋白基因在阿维链霉菌中的表达

EXPRESSION OF VITREOSCILLA HEMOGLOBIN GENE IN STREPTOMYCES AVERMITILIS

将含有自身启动子的透明颤菌血红蛋白基因（ ｖｈｂ） 克隆至大肠杆菌—链霉菌穿梭质粒载体ｐＩＪ６５３ 中构建成表达载体ｐ ＷＹ１０１ 和ｐ ＷＹ１０２ ，用它们转化阿维菌素（ａｖｅｒｍｅｃｔｉｎｓ） 产生菌———阿维链霉菌（ Ｓｔｒｅｐｔｏｍｙｃｅｓ ａｖｅｒｍｉｔｉｌｉｓ） ，经Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ 分析并未检测到ｖｈｂ 基因表达，但用穿梭载体ｐＨＺ１２５２（ 其中的ｖｈｂ 基因位于受硫链丝菌素诱导的链霉菌强启动子ＰｔｉｐＡ之下） 转化阿维链霉菌并经硫链丝菌素诱导，则在该菌中表达出了有活性的ＶＨｂ 蛋白。ｐＨＺ１２５２ 在阿维链霉菌中发生了重组缺失，但缺失的ｐＨＺ１２５２ 上仍含有完整的ｖｈｂ 基因及诱导型强启动子，且可在阿维链霉菌中稳定遗传，却不能再转化大肠杆菌。

Two expression vectors, pWY101 and pWY102, were constructed by cloning \%Vitreoscilla\% hemoglobin gene( \%vhb\%) with its native oxygen\|regulated promoter into \%E.coli\|Streptomyces\% shuttle vector pIJ653. They were introduced into \%Streptomyces avermitilis,\% but Western blotting experiment failed to detect \%vhb\% gene expression. pHZ1252 is another shuttle vector for expressing VHb, in which \%vhb\% structural gene is controlled by a strong, thiostrepton\|inducible \%Streptomyces\% promoter P\-\{\%tipA\%\}. pHZ1252 was transformed into \%S.avermitilis\% and expressed VHb which had biological activity after thiostrepton induction. pHZ1252 was structurally unstable in \%S.avermitilis\%, occurring deletion recombination. However, the rest part of pHZ1252 was stable in \%S.avermitilis\% and still contained \%vhb\% gene and P\-\{\%tipA\%\}. Plasmid pHZ1252 isolated from \%S.avermitilis\% was unable to transform \%E.coli,\% showing the loss of part of \%E.coli\% plasmid from pHZ1252.