人GPR81-Gi1α融合蛋白在杆状病毒系统中的表达及优化  

Fusion of Human GPR81 with Gila and its Expression with Baculovirus System

作　　者：吴芳明[1,2] 韩春光[1] 彭明丽[1] 黄琳仪[1] 王琼[1] 朱欣凯[1,2] 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 [2]江西中医学院中药系,南昌330004

出　　处：《生物技术通报》2008年第S1期149-154,共6页Biotechnology Bulletin

基　　金：国家自然科学基金(NO.30171096);“十五”全军医药卫生基金(No.01MB056)资助项目

摘　　要：旨在建立获得融合蛋白GPR81-Gi1α的方法和最优条件。采用RT-PCR从人胚胎肾及大脑组织总RNA中分别扩增孤儿G蛋白偶联受体GPR81和Gi1α的完整表达序列(分别为1 041bp和1 065bp),并构建各自的重组质粒pcDNA3.1(+)-GPR81及pcDNA3.1(+)-Gi1α,再以重组质粒为模板,运用重叠延伸PCR法扩增得到融合基因GPR81-Gi1α,测序无误后将融合基因与pFASTBac1重组得重组质粒pFASTBac1-GPR81-Gi1α,而后转化DH10Bac,使该融合基因重组质粒发生特异性的转座和病毒重组,获得杆状病毒表达穿梭质粒pBacmid-GPR81- Ci1α,再将该重组杆粒转染昆虫sf9细胞,获得含杆状病毒的细胞分泌上清,以上清在不同条件下(包括不同感染时间、滴度等)感染sf9细胞以优化融合蛋白在昆虫sf9细胞的表达条件。结果表明,感染72h且感染强度moi为5时是融合蛋白在sf9细胞中高效表达的理想条件。该表达体系的建立及蛋白表达条件的优化,保证了足量GPR81- Gi1α融合蛋白的制备。Our research aimed at obtaining the optimal condition for fusion protein GPR81-Gi1α.The whole ORFs of GPR81 and Gi1α(1041bp and 1065bp)were cloned by RT-PCR from total RNA of human fetus kidney and brain,respectively. Then,their corresponding recombinant plasmid pcDNA3.1(+)-GPR81 and pcDNA3.1(+)-Gi1αwere constructed as templates to splice GPR81 and Gi1αinto GPR81-Gilcdusion gene by overlap extension PCR method.After sequencing,the fusion gene was inserted into plasmid pFASTBacl to obtain the recombinant pFASTBac1-GPR81-Gi1α, from which pBacmid-GPR81-Gi1αwas successfully constructed with Bac-to-Bac baculovirus expression system indicated by specific transposition and virus recombination.With pBacmid-GPR81-Gi1α,the insect sf9 cells were transfected and the baculovirus carrying GPR81-Gi1αfusion gene in the culture supernatant was obtained.To optimize the expression condition of GPR81-Gi1α,sf9 cells were then infected with the baculovirus at different time(24～96h)and multiplicity of infection(1～10 moi).In this experiment,72h after infection with 5 moi of baculovirus was the optimal condition for expression of the fusion gene.The establishment of the GPR81-Gi1αexpression system with optimized condition will be useful for producing enough protein preparation.

关 键 词：GPR81 Gi1α 融合蛋白 bac-to-bac杆状病毒表达 

分 类 号：Q78[生物学—分子生物学]