摘要
本文应用电转移法将CD4、CD19抗原基因转入Cos7细胞获得高效转移和暂短表达,检测96h抗原阳性频率分别为3.7×10^(-3)和2.6×10^(-3)。将含有人IL-2RcDNA(CD25)的逆转录病毒载体pN-TK-IL-2R转染PA317和2病毒包装细胞系,收集病毒上清感染NIH3T3细胞;病毒滴度可至3.3×10~5CFU/ml,细胞传代40次间接免疫荧光及APAAP法均可见阳性转染细胞;RNA点杂交检测转染细胞有IL-2R基因mRNA转录产物表达。应用PCR方法扩增出逆转录病毒载体上选择标记基因(Neo),结果表明已获得稳定表达人IL-2R基因的转染细胞株。上述基因的转移和表达为有关抗原功能研究、鉴定及筛选相应单克隆抗体提供了有效的途径。
Human leukocyte differentiation antigens CD4 and CD19 were transfected into Cos cell line by electroporation. Indirect immunofluorescence and APAAP immunocytochemical staining shows that transient expression and high efficiency transformation of the two genes were obtained,we used pNTK-EL-2R,a retro-viral vector containing the human IL-2 receptor gene (CD25),to transfect amphotropic and ecotropic virus packing cell lines, subsequently .cell-free culture supernatant was used to infect murine fibroblast NTH3T3.. The liter of virus generated from virus-producing cells about 3. 3X 10s CPU/ml. IL-2R mRNA expression of transfected cells was assayed with Northern blot. Expression of the neo gene in retraviral vector was shown by PCR. These date demonstrate efficient transfer and expression of human IL-2R gene. The research provide a new approach for study of molecular structure and function of antigens,and of idendification and screening of McAbs.
出处
《免疫学杂志》
CAS
CSCD
北大核心
1993年第3期145-151,共7页
Immunological Journal
关键词
白细胞
分化抗原
受体
基因表达
Leukocyte differentiation antigen,Retroviral vector, IL-2 receptor, Gene expression
