烟实夜蛾信息素结合蛋白3 cDNA的克隆、序列分析与原核表达

Cloning and sequencing of cDNA encoding pheromone binding protein 3 from the Helicoverpa assulta (Guenée) and its expression in Escherichia coli

利用RT-PCR技术从烟实夜蛾Helicoverpa assulta( Hass)雄虫触角中扩增得到了信息素结合蛋白3( HassPBP3)。克隆和测序结果表明,该基因核苷酸序列全长495 bp,编码164个氨基酸残基,预测分子量18.5 kD。并预测N-末端疏水区包含由22个氨基酸组成的信号肽。因此,成熟蛋白应包括142个氨基酸,预测分子量为16.1 kD,等电点为5.44。经氨基酸序列同源性分析发现,此序列与已知昆虫PBP3有较高的同源性,而且具有气味结合蛋白的典型特征。将该基因重组到表达载体pGEX-4T-2中进行原核表达。经IPTG诱导、SDS-PAGE分析和Western印迹检测,结果表明烟实夜蛾PBP3基因能在大肠杆菌BL21中表达,电泳检测到一条大约42 kD的外源蛋白,与预测的融合蛋白分子量相符。

A cDNA clone encoding a pheromone binding protein 3 from antenna of Helicoverpa assulta （named HassPBP3） was isolated by reverse transcription polymerase chain reaction （RT-PCR）. The cloning and sequencing results showed that the full length of Hass PBP3 open reading frame （ORF） was 495 bp, encoding 164 amino acid residues, and the predicted molecular weight （MW） was 18.5 kD. The N-terminus hydrophobic region predicted containing of 22 amino acid residues within the HassPBP3 displayed the characteristic features of a signal peptide. Thus, the mature protein should consist of 142 amino acids with a calculated molecular weight （MW） of 16.1 kD and isoelectric point （IP） of 5.44. The gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by IPTG, the PBP3 proteins in H. assulta was expressed in Escherichia coli BL21, and its MW was found to be about 42 kD by checking with SDS-PAGE, nearly equal to the predicted.