cDNA Cloning,Prokaryotic Expression and Polyclonal Antibody Preparation of GOBP2 from Ostrinia furnacalis (Guenée)

【Objective】The objective of this study is to clone and express in prokaryotic system of a novel cDNA,named OfurGOBP2,encoding the general odorant binding protein GOBP2 from Ostrinia furnacalis(Guenee).【Method】The cDNA encoding OfurGOBP2 was isolated from Ostrinia furnacalis(Guenee) antennae by using reverse transcription-polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE).The open reading frame(ORF) of OfurGOBP2 was further cloned into prokaryotic cells to test its expression.【Result】 Sequencing and structural analysis showed that the ORF of OfurGOBP2 was 489 bp in size,encoding 162 amino acid residues(GenBank accession no.DQ673101).GOBP2 contains six conserved cysteine residues,consistent with the characteristics of odorant binding protein.The homologue analysis revealed that Ostrinia furnacalis GOBP2 shared 70% identity with other insects GOPB2,which indicated that insect GOPB2 is conserved in evolutional process.GOBP2 was further ligated with pGEX-4T-2 vector and then transformed into Escherichia coli BL21(DE3).SDS-PAGE and Western-blot results revealed that GOBP2 was expressed in E.coli.The molecular weight of expressed protein was consistent with the predicted molecular weight of OfurGOBP2.Anti-GOPB2 antibody specifically recognized OfurGOBP2 from antenna by using Western blot.【Conclusion】In this study,OfurGOBP2 was cloned and expressed in prokaryotic expression system,and the polyclonal antibody was further prepared,which is helpful for further researches on molecular structure and function of OfurGOBP2.