人oGPCRs与Gi1α的基因融合及其在昆虫细胞Sf9的表达  

Fusion of human orphan G protein-coupled receptors GPR45,GPR85 or GPR174 with Gi1α and their expression in insect Sf9 cells

作　　者：彭明丽[1] 韩春光[1] 高志清[1] 王琼[1] 高月[1] 刘永学[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《军事医学科学院院刊》2010年第1期8-11,共4页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的获得人孤儿G蛋白偶联受体(oGPCR)与G蛋白α亚基(Gi1α)的融合基因及其表达蛋白。方法采用RT-PCR法,从人HepG2细胞扩增oGPCR成员GPR45,GPR85,GPR174和Gi1α的完整表达序列,运用重叠延伸PCR法扩增得到人oGPCR成员GPR45,GPR85,GPR174分别与Gi1α的融合基因,将各融合基因与供体质粒pFASTBac1重组,而后分别转化DH10Bac菌株获得杆状病毒穿梭杆粒,制备重组杆状病毒并感染Sf9细胞,收获融合蛋白,经Western印迹鉴定。结果得到了上述oGPCR成员的融合基因oGPCRs-Gi1α,并使之在昆虫细胞成功表达,制备了足量的oGPCRs-Gi1α融合蛋白。结论oGPCR可与Gi1α成功融合,昆虫细胞表达体系是制备融合蛋白的理想手段,为今后深入开展oGPCRs的相关研究提供了保障。Objective To obtain the fusion genes of several human orphan G protein coupled receptors （oGPCRs） with Gi1α subtype of G protein and their expression system.Methods The whole open reading frames of GPR45,GPR85,GPR174 and Gilα were cloned by RT-PCR from HepG2 cDNA separately,and the corresponding fusion genes were amplified by overlap extension PCR.Then,the fusion genes-containing pBacmids were successfully constructed with the Bac-to-Bac baculovirus expression system indicated by specific transposition and virus recombination.The insect Sf9 cells were transfected with pBacmid-oGPCRs-Gi1α,and the supernatant containing recombinant virus was harvested.With the supernatant,insect Sf9 cells were infected under an optimized condition （MOI=5,infection time=72 h） and the fusion proteins were prepared and detected by Western blotting.Results The three fusion genes of GPCR45,GPR85 or GPR174 with Gi1α were obtained.The corresponding fusion proteins could be properly prepared in Sf9 cells.Conclusion Human oGPCRs could be fused with Gilα,and the fusion genes could be expressed using the Bac-to-Bac baculovirus expression system in insect Sf9 cells.

关 键 词：孤儿G蛋白偶联受体 G蛋白α亚基 融合蛋白 质粒 杆状病毒 表达系统 转染 

分 类 号：Q78[生物学—分子生物学]