人源KOR-GFP融合蛋白在昆虫细胞中的表达和活性鉴定  

Expression and Activity Identify of Human KOR-GFP Fusion Protein in Insect Cell

作　　者：周国超 石童 陈学军 王陈 李丽琴 张瑞华 鹿晓晶 

机构地区：[1]国民核生化灾害防护国家重点实验室,北京102205

出　　处：《药物生物技术》2017年第1期11-15,共5页Pharmaceutical Biotechnology

摘　　要：通过PCR方法扩增人源kappa型阿片受体基因(hKOR)和GFP基因,然后采用重叠延伸PCR(SOEPCR)将hKOR与GFP基因连接,并将融合基因通过无缝克隆技术克隆入pFastBac1载体,将筛选的阳性重组载体转化至含有杆状病毒穿梭载体和辅助载体的DH10αBac感受态细胞获得重组杆粒Bacmid-hKOR-GFP。采用重组杆粒转染Sf9昆虫细胞,收获上清作为重组病毒P1代,采用流式细胞术检测病毒感染效率;采用Western blot鉴定hKOR-GFP的表达情况;采用cAMP酶联免疫方法检测hKOR-GFP的功能活性。结果表明,采用感染复数(MOI)值为2,感染后72 h,hKOR-GFP在Sf9昆虫细胞中可达到高效表达;Western blot实验表明hKOR-GFP在昆虫细胞中获得正确表达;cAMP酶联免疫实验结果证明在激动剂存在下,Sf9细胞内cAMP含量出现剂量依赖性下降,表明hKOR-GFP具有生物活性。在昆虫细胞中高效表达具有功能活性的hKOR-GFP可为建立非成瘾性镇痛药物筛选技术平台奠定基础。The human kappa receptor（hKOR） and GFP genes were amplified by polymerase chain reaction（PCR）,and the hKORGFP fusion gene was amplified by overlap extension PCR method. After sequencing,the hKOR-GFP fusion gene was inserted into plasmid pFastBac1 by In-fusion cloning to obtain the recombinant pFastBac-hKOR-GFP,which was transformed into E. coli competent cells DH10αBac containing baculovirus shuttle vector and helper vector to obtain the recombinant Bacmid-hKOR-GFP. The recombinant Bacmid-hKOR-GFP was screened and verified by PCR. The insect Sf9 cells were transfected with Bacmid-hKOR-GFP and the baculovirus carrying hKOR-GFP fusion gene in the culture supernatant was obtained. To optimize the expression condition of hKOR-GFP,Sf9 cells were infected with the baculovirus at different time with multiplicity of infection of 2,and the infect efficiency was detected by flow cytometry. Western blot and cAMP enzyme immunoassay were performed to verify the expression and function of the recombinant protein. The result of flow cytometry showed that the optimal condition for expression of the fusion protein was 72 h after infection with MOI of 2. The result of Western blot showed that the recombinant protein hKOR-GFP was expressed correctly in Sf9 cells. The results showed that the cAMP level in Sf9 cells was inhibited in dose-dependent manner after being treated with agonist through cAMP enzyme immunoassay method. The experiments confirmed that the recombinant protein hKOR-GFP was functional expressed in Sf9 cells. Quantities of function fusion protein hKOR-GFP were expressed by insect baculovirus expression vector system（IBEVS）,which was greatly helpful for the selection of non-additional analgesic drugs.

关 键 词：杆状病毒表达系统 kappa阿片受体 流式细胞术 绿色荧光蛋白 转染效率 感染复数 

分 类 号：Q7[生物学—分子生物学]