苏云金芽孢杆菌cry1Ac28基因的克隆及原核表达

Cloning and procaryotic expression of cry1Ac28 gene from Bacillus thuringiensis

研究旨在克隆苏云金芽胞杆菌(Bacillus thuringiensis,Bt)Q-12的cry1Ac28基因并在原核载体中表达。应用PCR-RFLP法鉴定出Bt Q-12中含有cry1Ac基因,根据已知的cry1Ac全长基因序列设计特异引物,以Bt Q-12基因组DNA为模板扩增cry1Ac全长基因,与大肠杆菌(Escherichia coli)表达载体pEB相连接获得含有cry1Ac全长基因的重组质粒pEB-cry1Ac,经过序列分析表明,该基因编码区为3 537 bp,编码1 178个氨基酸,分子质量为133.3176 ku,pI为4.885,为弱酸性蛋白质,亮氨酸(Leu)、丝氨酸(Ser)、谷氨酸(Glu)三种氨基酸含量最高,分别为8.06%、7.80%、7.72%,该基因在大肠杆菌BL21(DE3)菌株能够正常表达133.3176 ku蛋白。该基因核苷酸序列已在GenBank中登录,登录号为FJ610439,并由Btδ-内毒素基因国际命名委员会正式命名为cry1Ac28。为进一步研究cry1Ac28蛋白功能和活性打下了良好的基础。

Bacillus thuringiensis Q-12 was identified to harbour cry1Ac gene by PCR-RFLP.According to the published sequence of cry1Ac gene,a pair of special primers was designed for full ength DNA cloning of cry1Ac gene by PCR amplification.Subsequently,the amplified fragment of cry1Ac gene was inserted into the procaryotic vector pEB and a 133.3176 ku peptide was expressed in Escherichi coli BL21（DE3） by IPTG induction.The gene was registered in GenBank with accession number FJ610439 and was designated as a novel gene,cry1Ac28,by International Nomenclature Committee of Bt.Sequence analysis results showed that the open reading frame of the cry1Ac gene was 3 537 bp which coded 1 178 amino acids and its pI was 4.885.In the amino,acids serine,leucine and threonine were the most rich,with the content of 8.06%,7.80% and 7.72%,respectively.These results provided the basis for the future research on cry1Ac28 protein function and activity.