摘要
【目的】探索建立一种有效制备昆虫非典型嗅觉受体Orco抗原的方法,为Orco蛋白组织定位及功能研究奠定基础。【方法】设计带有Bam H I和Hind III酶切位点的引物,采用RT-PCR方法扩增家蚕Bombyx mori Orco第4-5跨膜区之间的基因片段,将其与原核表达载体p ET-28a(+)双酶切处理后进行连接,然后转化大肠杆菌Escherichia coli感受态细胞DH5α,重组质粒再转化大肠杆菌BL21(DE3)菌株,用IPTG诱导表达,用SDS-PAGE检测诱导蛋白,用His Trap HP亲和层析对诱导表达的大量蛋白进行纯化。【结果】在大肠杆菌原核表达系统中,用IPTG诱导获得了与预测蛋白大小相符的目的蛋白,经SDS-PAGE检测发现目的蛋白以包涵体形式表达,在变性条件下经His Trap HP亲和层析获得大量可用于抗体制备的纯化蛋白。【结论】利用原核表达系统可获得制备家蚕Orco抗体的抗原蛋白。
[Aim]To establish an effective method to prepare the antigen protein of the atypical insect olfactory receptor Orco,and to provide the foundation for study on the immunolocalization and function of Orco. [Methods]The target fragment between the 4th- 5th transmembrane regions of Orco gene was amplified from the silkworm,Bombyx mori,by RT-PCR using primers containing restriction sites of Bam H I and Hind III. This target fragment was digested with Bam H I and Hind III directly and ligated to the p ET-28a（ ＋） expression vector digested with the same two restrictive enzymes. The ligated product was transformed into the competent Escherichia coli cell DH5α,and the positive clones were testified by PCR and sequenced. The right recombinant expression plasmids were transformed into BL21（ DE3）strains,which then were induced with IPTG. SDS-PAGE was used to identify the expressed recombinant protein. A large amount of the induced protein was purified by His Trap HP affinity chromatography.[Results]The target protein whose size was consistent with the predicted protein was induced in E. coli prokaryotic expression system. The target protein was expressed in the form of inclusion body detected by SDS-PAGE. A large amount of purified protein which can be used for antibody preparation was obtained by His Trap HP affinity chromatography under denaturing conditions. [Conclusion]The antigen protein used for antibody preparation of silkworm olfactory receptor Orco can be obtained by prokaryotic expression system.
出处
《昆虫学报》
CAS
CSCD
北大核心
2015年第11期1194-1200,共7页
Acta Entomologica Sinica
基金
国家重点基础研究发展计划("973"计划)项目(2012CB114603)
山东科技发展计划项目(2014GNC110001)
现代农业产业技术体系建设专项基金项目(CARS-22-ZJ0302)
山东农业大学博士后基金项目(76364)
山东省现代农业产业技术体系蚕桑产业创新团队建设项目(31892)
关键词
家蚕
原核表达
非典型嗅觉受体
Orco
抗原蛋白
Bombyx mori
prokaryotic expression
atypical olfactory receptor
Orco
antigen protein
