棉铃虫普通气味结合蛋白Ⅱ基因的表达及鉴定

Expression and identification of general odorant binding protein Ⅱ from Helicoverpa armigera (Hübner)

通过PCR扩增的方法获得了棉铃虫普通气味结合蛋白Ⅱ (GOBP2 Harm)基因成熟蛋白阅读框序列 ,构建了GOBP2 Harm原核表达载体pGEX GOBP2 Harm ,并成功地在大肠杆菌中进行了表达。SDS PAGE分析表明 :大部分GOBP2 Harm重组蛋白形成不溶性的包涵体 ,超声波破碎大肠杆菌细胞后 ,在上清液中能检测到少量的可溶性GOBP2 Harm蛋白。为了获得大量纯化的可溶性目的蛋白 ,我们对包涵体进行了溶解和重折叠 ,并通过亲合层析法进行了纯化。纯化产物能与多音天蚕(Antheraeapolyphemus)GOBP抗血清发生交叉反应 ,证实表达产物属于昆虫普通气味结合蛋白。

The intact open-reading fragment sequence of mature GOBP2-Harm from Helicoverpa armigera was obtained by PCR amplification. The ORF of GOBP2-Harm was subcloned into expression vector pGEX-4T-1. Recombinant protein was expressed successfully in E.coli induced by IPTG. The analysis of SDS-PAGE showed that most of recombinant protein was insoluble inclusion body and only little fraction of recombinant protein of GOBP2-Harm is soluble. In order to obtain abundant soluble recombinant protein, the insoluble inclusion body GOBP2-Harm was solubilized, refolded and purified. The purified product was crossreactive with an anti-GOBP (Antheraea polyphemus) antiserum, which indicated the purified protein belonged to GOBP of insect.