【摘要】 利用RT PCR技术扩增了编码烟实夜蛾Helicoverpaassulta雌、雄虫触角普通气味结合蛋白Ⅱ的cDNA片段 ,将其克隆至pGEM TEasy载体 ,获得了普通气味结合蛋白Ⅱ基因成熟蛋白阅读框序列。将该基因重组到表达型质粒pET 30a(+)中 ,并转化入原核细胞中表达。序列测定结果表明 ,烟实夜蛾触角普通气味结合蛋白基因的成熟蛋白阅读框全长 4 89bp ,编码 16 2个氨基酸残基 ,预测分子量和等电点分别为 18 2kD和 5 35。推导的氨基酸序列与已报道的 10种昆虫普通气味结合蛋白Ⅱ高度同源 (73%～ 98% ) ,并具有气味结合蛋白的典型特征。SDS PAGE和Western印迹分析表明 ,经IPTG诱导 ,普通气味结合蛋白Ⅱ基因能在大肠杆菌BL2 1(DE3)中表达 ,电泳检测到一条约 2 3kD大小的外源蛋白 ,与预测的融合蛋白分子量大小相应。更多还原

【Abstract】 The cDNA encoding the general odorant binding protein Ⅱ (named as Hass-GOBP2) was isolated from the male and female antennae of Helicoverpa assulta (Guenée) by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragment was further cloned into pGEM-T Easy vector, and then constructed into expression vector pET-30a(+) for overexpression in prokaryotic cells. Structural analysis showed that the full length of mature Hass-GOBP2 open reading frame (ORF) was 489 bp, encoding 162 amino acid residues; the predicted MW and pI were 18.2 kD and 5.35, respectively. The deduced amino acid sequence showed a high identity to the reported sequences of GOBP2 from other insects and shared the typical structural features of odorant binding proteins from other insects. Induced by IPTG, the full length of GOBP2 was expressed in Escherichia coli BL21 (DE3). Its molecular weight was found to be about 23 kD by checking with SDS polyacrylamide gel electrophoresis and Western blotting.更多还原