通过克隆cry1Ac基因BtⅠ-BtⅡ启动子、SD序列以及终止子,并引入pET28a的His标签和多克隆位点,在穿梭载体pHT304的基础上构建了一个苏云金芽孢杆菌表达载体pBMB1A.将cry1Ac以及具有非典型BtⅠ-BtⅡ启动子的cry2Ab、cry5Ba、cry6Aa、cry7Ba、cry55Aa6个基因装载到该表达载体上,转入BMB171构建了重组菌株,通过复红简单染色后的显微镜镜检结果表明,重组菌株BMB1A-1Ac、BMB1A-5Ba、BMB1A-7Ba和BMB1A-55Aa均能形成正常晶体,SDS-PAGE结果也证实这4个重组菌株的重组质粒均能表达出目的蛋白.同时,选取重组菌株BMB1A-1Ac来考察His标签对Cry1Ac杀虫活性的影响,生物活性测定结果显示重组菌株的LC50值与对照菌株相比无明显差异.该载体可用于快速克隆表达不同类别的Cry蛋白.%A Bacillus thuringiensis expression vector named Pbmbia was constructed by cloning BtI-BtII promoter, SD-sequence, and transcription terminator of cry 1 Ac gene, and adding His-tag and the following multiple cloning sites (MCS) from pET28a into the shuttle vector Pht304. Cry 1 Ac and other five cry genes with atypical Btl-Btll promoter including cry2Ab, cry5Ba, cry6Aa, cry7Ba, cry55Aa were cloned into Pbmbia. Microscopic observation showed that the recombinant strains containing CrylAc, Cry5Ba, Cry7Ba and Cry55Aa could form parasporal inclusion bodies; And SDS-PAGE proved that all of them could produce the corresponding major insecticidal crystal proteins bands. Meanwhile, CrylAc was selected to determine the effect of His-tag on its insecticidal activity, and the bioassay result showed that there was no significant difference between the LC50 of the recombinant strain and the control strain. The expression vector constructed was suitable for rapid cloning and expression of different cry genes.
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