摘要
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.
出处
《微生物学报》
CAS
CSCD
北大核心
2000年第1期50-56,共7页
Acta Microbiologica Sinica
关键词
透明颤菌
血红蛋白
阿维链霉菌
VHB
基因表达
Vitreoscilla hemoglobin (VHb), Streptomyces avermitilis, vhb gene expression
