苏云金芽孢杆菌δ-内毒素cryIA(c)基因在大肠杆菌和变铅青链霉菌中表达

Expression of δendotoxin cryIA(c) Gene of Bacillus thuringiensis in Escherichia coli and Streptomyces lividans

利用合成的寡聚核苷酸片段，从质粒ｐＯＳ１０００中分离出具有适合克隆位点的苏云金芽孢杆菌（Ｂａｃｉｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ）δ内毒素ｃｒｙＩＡ（ｃ）全长基因。将该基因与大肠杆菌表达载体ｐＫＫ２２３３重组，并引入大肠杆菌ＪＭ１０９中，经ＩＰＴＧ诱导，获得了超量表达的ＣｒｙＩＡ（ｃ）蛋白。将ｃｒｙＩＡ（ｃ）全长基因插入链霉菌表达载体ｐＨＺ１２７２中，得到重组质粒ｐＨＺ１２５６，将该质粒引入变铅青链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓｌｉｖｉｄａｎｓ）ＪＴ４６中，经硫链丝菌素诱导，通过Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ测定表明，重组变铅青链霉菌ＪＴ４６（ｐＨＺ１２５６）已表达出相应的ＣｒｙＩＡ（ｃ）蛋白。杀虫试验表明，大肠杆菌和链霉菌所表达出的δ内毒素ＣｒｙＩＡ（ｃ）对小菜蛾均有毒杀作用，其致死率分别为９３％和５７％。

The cryIA(c) gene of Bacillus thuringiensis was isolated from plasmid pOS1000.In order to obtain a proper cloning site and open reading frame,some DNA sequences preceeding the initiating codon of the gene were replaced by synthetic oligonucleotide sequences.The isolate cryIA(c) was cloned into E.coli expression vector pKK223 3,and the production of CryIA(c) protein was detected after induction by IPTG.A recombinant plasmid,pHZ1256,was constructed by insertion of the cryIA(c) gene into Streptomyces vector pHZ1272.pHZ1256 was introduced into Streptomyces lividans,and the production of CryIA(c) protein was confirmed by Western blotting after thiostrepton induction.Bioassy experiment showed that the CryIA(c) protein produced by E.coli and S.lividans caused 93% and 57% mortality to Plutella xylostella,respectively.