亚洲玉米螟GOBP2的克隆、原核表达及多克隆抗体制备

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

【目的】克隆、序列分析和原核表达亚洲玉米螟(Ostrinia furnacalis)GOBP2(OfurGOBP2)的cDNA。【方法】以亚洲玉米螟触角为材料,采用RT-PCR结合RACE方法克隆OfurGOBP2的cDNA序列,并在pGEX-4T-2/BL21(DE3)系统中进行原核表达,进一步利用制备的抗体检测OfurGOBP2蛋白。【结果】从亚洲玉米螟触角中获得了GOBP2的cDNA序列(GenBank登录号为DQ673101),序列分析表明,OfurGOBP2开放阅读框489 bp,编码162个氨基酸残基,氨基酸序列中有6个保守的半胱氨酸位点,具有气味结合蛋白的典型特征。一致性分析显示,OfurGOBP2与其它鳞翅目昆虫GOBP2编码的氨基酸一致性较高,表明昆虫的GOBP2在分子进化过程中是保守的。进一步将OfurGOBP2与表达载体pGEX-4T-2连接,转入大肠杆菌BL21(DE3)中,经IPTG诱导成功表达了相对分子质量为41 kD的可溶性融合蛋白。SDS-PAGE分析和Western印迹检测结果表明,OfurGOBP2能够高效表达,并与预测的融合蛋白分子量相符。利用制备的多克隆抗体对亚洲玉米螟GOBP2进行Western-blot分析,证明其能够特异识别OfurGOBP2蛋白。【结论】成功克隆了编码并表达亚洲玉米螟气味结合蛋白GOBP2的cDNA序列,并制备了多克隆抗体,可用于深入研究亚洲玉米螟GOBP2的结构和功能。

【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（Guenée）.【Method】The cDNA encoding OfurGOBP2 was isolated from Ostrinia furnacalis（Guenée） 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.