苏云金芽孢杆菌分子伴侣串联基因p19-p29的克隆和表达载体的构建

Cloning of the Molecular Chaperone Gene p19-p29 from Btand Construction of the Bt Expression Vector

根据已知序列设计一对PCR引物 (ORF 5S ,ORF 3N) ,可从cry2Aa或cry2Ac操纵子中扩增出包含串联分子伴侣基因p1 9 p2 9的DNA片段 ,预期大小分别为 1 6kb和 2 0kb。对 1 5 0株苏云金芽孢杆菌菌株进行PCR检测 ,从 2 6株中获得了大小为 1 6kb的扩增片段 ,但未获得2 0kb的片段。这表明cry2Aa型操纵子p1 9 p2 9基因存在较广泛 ,而cry2Ac型较罕见。将来自Y2 菌株的 1 6kb片段回收 ,通过一系列亚克隆 ,最终构建成一个含有p1 9 p2 9串联基因的Bt表达载体 ,为进一步研究p1 9 p2

The primer pair (ORF-5S,ORF-3N)designed according to the known sequence can amplify a DNA fragment, containing molecular chaperone genes p19 and p29, in size of 1.6kb from the cry2Aa operon or of 2.0kb from the cry2Ac operon. 150 Bacillus thuringiensis(Bt) strains were detected by PCR with the primer pair. With a blankness of the 2.0kb fragment, the expected 1.6kb fragment was acquired from 26 Bt strains. These results showed that the genotype of cry2Aa operon widely existed in Bt and that cry2Ac was rare. The 1.6kb PCR product from strain Y 2 was recovered. After several steps of sub-cloning, it was at last inserted into the shuttle vector pHT3101, resulting in an expression vector pHY 2P with multiple clone sites.