关键词: 斜纹夜蛾, 触角, 气味结合蛋白, 基因克隆, 原核表达

Abstract:

【Objective】 The objective of this study aims at cloning, sequence analysis and prokaryotic expression of a new cDNA encoding the general odorant binding protein Ⅰ from Spodoptera litura (named as SlGOBPⅠ). 【Method】 The cDNA encoding SlGOBPⅠ was isolated from the antennae of S. litura (SlGOBPⅠ, GenBank Accession No. EU086372) by homology cloning and rapid amplification of cDNA ends (RACE), and then the open reading frame (ORF) of SlGOBPⅠ was cloned into prokaryotic cells to test its expression. 【Result】Results of sequencing and structural analysis showed that the ORF of SlGOBPⅠ was 495 bp, encoding 164 amino acid residues, with the predicted MW of 19.3 kD and pI of 5.54, respectively. It shared the typical structural features of odorant binding proteins from other insects, including the six conservative Cys lotus in the sequence. The deduced amino acid sequence showed a high identity to the reported sequences of GOBPⅠ from other lepidopteran insects, and showed that SlGOBPⅠ may belong to the family of GOBP. The SlGOBPⅠ was constructed into pET-32a vector and expressed in Escherichia coli BL21 (DE3) after induction with IPTG. The analysis of SDS polyacrylamide gel electrophoresis and Western blot indicated that the molecular weight of fusion protein was about 32.0 kD. The fusion protein was purified by one step Ni-NTA affinity chromatograph column and injected into New Zealand White rabbit to raise antiserum successfully. The analysis of ELISA showed the titer of antiserum was 1﹕5 000. Western blot analysis showed that the expressed SlGOBPⅠ protein was crossreactive with the anti-SlGOBPⅠ antiserum, which indicated the expressed fusion protein belonged to GOBPⅠ of the insect. 【Conclusion】The successful cloning and expression of the coding sequence of SlGOBPⅠhave laid a basis for further study on the structure and function of SlGOBPⅠ.

Key words: Spodoptera litura, antennae, general odorant binding proteinⅠ, gene cloning, prokaryotic expression