论文部分内容阅读
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。
OBJECTIVE: To clone human CXCR3B gene and construct eukaryotic expression vector containing the target gene to obtain the gene transfection cell line L929-huCXCR3B stably expressing human CXCR3B molecule. Methods: The human CXCR3B gene was amplified from pMD19-T / huCXCR3A plasmid by PCR and inserted into the eukaryotic expression vector pIRES2-EGFP by double enzyme digestion. The L929 cells were transfected by lipofectamine and the positive clones were screened by G418. The expression of human CXCR3B mRNA and protein in positive clones was analyzed by RT-PCR and immunofluorescence respectively. MTT analysis of gene-transfected cell line L929-huCXCR3B in Mig (monokineinduced by IFN-γ, IFN-γ induced monocyte factor) under the action of proliferation. Results: The human CXCR3B gene was successfully cloned and the eukaryotic expression vector pIRES2-EGFP / huCXCR3B was constructed. After transfected with the vector, the gene-transfected cell line L929-huCXCR3B stably expressing human CXCR3B was obtained, and the positive expression of CXCR3B on the membrane surface The expression rate was 93%. The gene-transfected cells were co-cultured with its ligand Mig for 24,48 and 72 h, respectively. The inhibition rates were 41.44%, 44.01% and 24.80% respectively. Conclusion: The establishment of L929-huCXCR3B cell line lays the foundation for the study of CXCR3B signal transduction and preparation of the corresponding monoclonal antibody (mAb).