人CXCR3B基因转染细胞株的构建、鉴定及生物学功能研究  

Construction of human CXCR3B gene transfected cell line,identification and study of its biological function

作　　者：郭静雅[1] 陈昌友[1] 王志强[1] 张彦军[1] 黄赛男[1] 朱华亭[1] 邱玉华[1] 

机构地区：[1]苏州大学医学部免疫学系,江苏苏州215123

出　　处：《细胞与分子免疫学杂志》2011年第12期1288-1290,1294,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家科技部重大专项资助(2009ZX09103-705)

摘　　要：目的:克隆人CXCR3B基因,并构建含有该目的基因的真核表达载体,获得稳定表达人CXCR3B分子的基因转染细胞株L929-huCXCR3B。方法:采用PCR方法从pMD19-T/huCXCR3A质粒中扩增人CXCR3B基因,通过双酶切装入真核表达载体pIRES2-EGFP中;脂质体法转染L929细胞,G418加压筛选阳性克隆;分别用RT-PCR方法与免疫荧光技术分析阳性克隆中人CXCR3B在mRNA和蛋白水平的表达。MTT分析基因转染细胞株L929-huCXCR3B在Mig(monokineinduced by IFN-γ,IFN-γ诱导的单核因子)作用下的增殖能力。结果:成功克隆了人CXCR3B基因并构建了真核表达载体pIRES2-EGFP/huCXCR3B,转染该载体后获得了稳定表达人CXCR3B的基因转染细胞株L929-huCXCR3B,膜表面CX-CR3B分子的阳性表达率为93%。该基因转染细胞与其配体Mig共培养24、48及72 h,抑制率分别为41.44%、44.01%和24.80%。结论:L929-huCXCR3B细胞株的建立为研究CXCR3B信号转导及制备相应的单克隆抗体(mAb)奠定了基础。AIM： To construct recombinant human CXCR3B gene expression vector and obtain L929-CXCR3B gene transfected cell line for stably expressing human CXCR3B. METI-IODS： Human CXCR3B gene of full length was amplified by PCR from the plasmid pMDI9-T/huCXCR3A. Then, it was inserted into eukaryotic expression vector plRES2-EGFP to construct recombinant vector plRES2- EGFP/huCXCR3B. The recombinant vector was transfected into L929 cells with LipofectamineTM 2000, and cell clones stably expressing human CXCR3B molecule were screened by G418. We used FCM and RT-PCR to verity expression of CXCR3B from protein level and gene level. The ability of proliferation of L.929-huCXCR3B under the circumstance of CXCP,3B ligand called Mig was analyzed via MTT methods. RESULTS： We have constructed recombinant human CXCR3B gene expression vector and obtained L929-huCXCR3B gene transfected cell line which can stably express human CXCR3B molecule. The positive expression rate of CXCR3B on L929-huCXCR3B cells was 93%. The result of MTT assay showed that the proliferation of L929-huCXCR3 B cells were inhibited when the cells were cocultured with Mig after 24 h, 48 h and 72 h , and the inhibition ratio were 41.44%, 44. 01% and 24. 80% respectively. CONCLUSION： Construction of L929-huCXCR3B cell line has laid a good foundation on research of the CXCR3B signal pathway and preparation of the CXCR3B monoclonal antibody.

关 键 词：CXCR3B 趋化因子 L929细胞 基因转染 

分 类 号：R392.11[医药卫生—免疫学]