烟实夜蛾触角化学感受蛋白cDNA的克隆、序列分析及在大肠杆菌中的表达

Cloning and sequencing of cDNA encoding a chemosensory protein in the antenna of Helicoverpa assulta and its expression in Escherichia coli

利用RT-PCR技术扩增了编码烟实夜蛾Helicoverpa assulta触角化学感受蛋白(chemosensory protein)的全长cDNA。克隆和测序结果表明,烟实夜蛾化学感受蛋白基因核苷酸序列全长384 bp(GenBank序列号: DQ285667) ,编码127个氨基酸残基,预测N-末端包含16个氨基酸组成的信号肽序列,因此估测其成熟蛋白分子量为12.97 kD,等电点为5.32。将该基因重组到表达载体pGEX-4T2中,并转入原核细胞中进行表达。SDS-PAGE和Western印迹分析表明,经IPTG诱导后,烟实夜蛾化学感受蛋白基因能在大肠杆菌BL21中表达,电泳检测到一条约39 kD的外源蛋白,与预测的融合蛋白分子量大小相符。

The full-length eDNA encoding a ehemosensory protein （CSP） was isolated from the antenna of Helicoverpa assulta by reverse transcription polymerase chain reaction （RT-PCR）. This PCR fragment was further cloned and sequenced. The results showed that the CSP gene in H. assulta was 384 bp in size （registered in GenBank with the accession number DQ285667） and encoded 127 amino acid residues. The N- terminus hydrophobie region predicted containing of 16 amino acid residues within the Has-CSP displayed the characteristic features of a signal peptide. Thus the predicted mature weight （MW） is 12.97 kD and isoeleetrie point （IP） 5.32. The CSP gene was then constructed into the expression vector pGEX-4T2 for overexpression in prokaryotie cells. The SDS-PAGE analysis indicated that the CSP gene was expressed in Escherichia coli BL21. The Western blot further confirmed this result. The expressed fusion protein in BL21 was found to be about 39 kD, consistent with the predicted result.