Molecular Cloning And Sequencing Of CDNA Encoding General Odorant Binding Protein 2 In The Antenna Of Helicoverpa Assulta (Guenée) And Its Expression In Escherichia Coli

Insect can perceive certain chemical volatiles in the environment as the chemical information sources for its behaviroral reactions such as to look for mate, food and reproduction place. Insect odorant binding proteins (OBPs) are thought to deliver odors to olfactory receptors, and thus may be the first biochemical step in odor reception capable of some level of odor discrimination. And it's important for insect to transfer odor information in the environment and give rise to certain behavior response. In this paper, the gene of general odorant binding protein 2 in the antenna of Helicoverpa assulta (Guenî–«)was cloned and expressed in Escherichia coli.After comparing the reported GOBP2 nucleotide sequences of insects, which was obtained from GenBank/EMBL/DDBJ, the primer was designed. The cDNA encoding the general odorant binding protein 2 (named as Hass-GOBP2) was isolated from the male and female antenna of Helicoverpa assulta Guen6e by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragment (the accession numberof Hassu-GOBP2 is AY351670 in GenBank) was further cloned into pGEM-T Easy vector, respectively. The aimed fragment was digested by two restriction enzyme BamHI/XhoI and then was joined with pET-30a (+) plasmid which was digested by BamHI/XhoI too. And then constructed into expression vector pET30a (+) for overexpression in prokaryotic cells. Structural analysis showed that the full length of mature Hass-GOBP2 open reading frame (ORF) is 489bp, 162 amino acid residues were encoded. Thepredicted MW and pI are 18.2kDa and 5.35, respectively. The deduced amino acid sequence showed a high identity to the reported sequence of GOBP2 from different species of insects and shared the typical structural features with odorant binding proteins from other insects. The phylogenetic tree also showed the amino acid of Helicoverpa assulta and Helicoverpa zea have high homology. The kinship of eleven species each other are different from the genetic relationship of the eleven amino acids. After induced by IPTG, the full length of GOBP2 was expressed in Escherichia coli BL21 (DE3). Its molecular weight was about 23kDa, by checking with SDS polyacrylamide gel electrophoresis and Western blotting.We integrated GOBP2 gene into procaryotic expression vector. The recombinant protein is integrated with His Tag that is a purification of this protein which establish foundation to research the structure and the affinity of ligand.