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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的表达

Expression of delta-endotoxin cryIA(c) gene of Bacillus thuringiensis in Escherichia coli and Streptomyces lividans.

作者信息

Yang R, Hu Z, Deng Z, Li J

机构信息

Huazhong Agricultural University, Wuhan, China.

出版信息

Chin J Biotechnol. 1998;14(2):59-65.

Abstract

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 preceding the initiating codon of the gene were replaced by synthetic oligonucleotide sequences. The isolated cryIA(c) was cloned into E. coli expression vector pKK223-3, and 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. A bioassy experiment showed that the CryIA(c) protein produced by E. coli and S. lividans caused 93% and 57% mortality to Plutella xylostella, respectively.

摘要

苏云金芽孢杆菌的cryIA(c)基因是从质粒pOS1000中分离出来的。为了获得合适的克隆位点和开放阅读框,该基因起始密码子之前的一些DNA序列被合成寡核苷酸序列所取代。将分离得到的cryIA(c)克隆到大肠杆菌表达载体pKK223-3中,经IPTG诱导后检测到CryIA(c)蛋白的产生。通过将cryIA(c)基因插入链霉菌载体pHZ1272构建了重组质粒pHZ1256。将pHZ1256导入变铅青链霉菌,经硫链丝菌素诱导后通过蛋白质免疫印迹法证实了CryIA(c)蛋白的产生。生物测定实验表明,大肠杆菌和变铅青链霉菌产生的CryIA(c)蛋白对小菜蛾的致死率分别为93%和57%。

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