中华蜜蜂OBP3基因的克隆、原核表达及组织表达谱  

Cloning,prokaryotic expression and tissue expression profiling of an OBP3 gene in the Chinese honeybee,Apis cerana cerana(Hymenoptera:Apidae)

作　　者：吉挺[1] 沈芳[1] 梁勤[2] 吴黎明[3] 刘振国[1] 罗岳雄[4] 

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009 [2]福建农林大学蜂学院,福州350002 [3]中国农业科学院蜜蜂研究所,北京100093 [4]广东省昆虫研究所,广州510260

出　　处：《昆虫学报》2014年第8期897-904,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31372382);国家蜂产业技术体系(CARS-45-SYZ6);江苏省科技支撑计划课题(SBE201230535)

摘　　要：【目的】气味结合蛋白质(odorant binding proteins,OBPs)参与气味分子的识别,在蜜蜂嗅觉中扮演重要的角色。本研究旨在克隆中华蜜蜂Apis cerana cerana OBP3基因,以制备多克隆抗体。【方法】运用RT-PCR技术从中华蜜蜂头部总RNA中扩增OBP3基因,将该基因亚克隆入原核表达载体pET-28a并转入大肠杆菌Escherichia coli Rosetta(DE3)中诱导表达获得融合蛋白质,融合蛋白质经纯化后免疫新西兰白兔制备多克隆抗体,最后分别用间接ELISA和Western Blot检测抗体的效价和特异性,并采用荧光定量PCR检测OBP3基因在中蜂不同组织中的表达。【结果】克隆得到了中华蜜蜂OBP3基因AccOBP3(GenBank登录号KJ026357),大小为444 bp。SDS-PAGE结果显示融合蛋白成功表达。制备的多克隆抗体效价高于1∶40 000,且具有很高的特异性。荧光定量PCR结果表明,AccOBP3基因在腿部和触角中显著高表达(P<0.01),胸部中次之(P<0.01),头部和腹部中显著低表达,后两者表达量差异不显著(P>0.05)。【结论】OBP3基因在中蜂触角有高转录活性。本研究实现了中蜂OBP3基因的原核表达,并制备了兔抗中蜂OBP3多克隆抗体,为深入研究中蜂OBP3基因的功能奠定基础。【Aim】Odorant binding proteins（OBPs） are involved in the identification of odor molecules,and play an important role in bees’ olfaction. This study was designed to clone and express an OBP3 gene from the Chinese honeybee Apis cerana cerana,in order to prepare the polyclonal antibody.【Methods】The OBP3 gene was amplified by RT-PCR from total RNA from head of A. cerana cerana,then sub-cloned into prokaryotic expression vector pET-28 a and expressed in Escherichia coli Rosetta（DE3） host cells. The fusion protein was purified and immunized into New Zealand white rabbits so as to prepare polyclonal antibody. The sensitivity and specificity of the polyclonal antibody were detected through indirect ELISA and Western blot,respectively. The expression profiles of AccOBP3 in different tissues were detected by real-time quantitative PCR. 【Results】 AccOBP3（ GenBank accession no.KJ026357） was cloned and a 444 bp fragment was obtained. The recombinant vector pET-OBP3 was successfully constructed. SDS-PAGE analysis showed that the fusion protein was well expressed. The anti-OBP3 polyclonal antibody showed high titer（ higher than 1 ∶ 40 000） and specificity. Real-time quantitative PCR results showed that the expression level of AccOBP3 gene was significantly higher in legs and antennae（ P 〈 0. 01）,moderate in thorax,and significantly lower in heads and abdomen（ P 〈0. 01）. The expression level of AccOBP3 in heads and abdomen had no significant difference（ P 〉0. 05）. 【Conclusion】The OBP3 gene has high transcript expression in the antennae of A. cerana cerana. In this study, AccOBP3 gene has been cloned and expressed, and the rabbit antiApisCeranaOBP3 polyclonal antibody has been prepared by immunization with the purified recombinant OBP3 protein,which lays the foundation for further studies on functions of OBP3 gene.

关 键 词：中华蜜蜂 OBP3基因 基因克隆 原核表达 组织表达谱 抗体制备 

分 类 号：Q966[生物学—昆虫学]