棉铃虫细胞色素P450 CYP6B7基因的克隆与融合表达

细胞色素P450 CYP6B7被推测与棉铃虫Helicoverpa armigera对拟除虫菊酯类杀虫剂的抗性有关，但至今尚无CYP6B7参与杀虫剂代谢方面的直接证据。为揭示CYP6B7的代谢功能，作者以棉铃虫幼虫基因组DNA 为模板，以CYP6B7基因设计特异性引物，扩增出包含321 bp内含子的CYP6B7基因。用反向PCR的方法消除内含子，获得包含完整的CYP6B7基因的开放阅读框。将CYP6B7基因与pMAL-c2X载体连接，并转化E.coli TB1细胞，在IPTG诱导下，CYP6B7能与载体基因编码的麦芽糖结合蛋白（MBP）在大肠杆菌中融合表达，表达产物经直链淀粉（amylose） 柱亲和层析分离洗脱后，得到SDS-PAGE电泳纯的融合蛋白。

Cloning and fusion expression of CYP6B7 gene from Helicoverpa armigera

Although it has been suggested that CYP6B7 in the cotton bollworm, Helicoverpa armigera, is probably responsible for resistance to pyrethroids, there is no direct evidence showing that CYP6B7 is involved in the metabolism of pyrethroid pesticides. To explore the function of CYP6B7 gene, we attempted to produce CYP6B7 enzyme through heterologous expression of the CYP6B7 gene. The open reading frame of CYP6B7 gene was isolated by PCR with genomic DNA as the template and using a pair of CYP6B7 gene specific primers, followed by reverse PCR aiming at deleting the intron sequence of 321 bp. A fusion plasmid (CYP6B7-pMAL) was constructed by inserting the CYP6B7 gene into the BamHⅠ-SalⅠ restriction sites of pMAL-c2X vector. CYP6B7 pMAL plasmid was transformed into E. coli TB1. A fusion protein (CYP6B7 fused with maltose binding protein) was expressed after induction by IPTG..SDS-PAGE pure fusion protein was isolated with an amylose column.