摘要
目的 :基因转移人转化生长因子β1(TGF β1)在真核细胞系中蛋白表达。方法 :借助真核质粒表达载体 ,将人TGF β1 转移入小鼠成纤维细胞株(NIH/3T3)细胞中 ,经G418筛选 ,克隆扩增 ,Southernblot、Northernblot、PCR、RT PCR鉴定 ,并经水貂肺上皮细胞(CCL 64)生长抑制法 ,对克隆细胞分泌TGF β1 蛋白进行活性检测。结果 :证实了重组人TGF β1 基因在NIH/3T3细胞基因组中的稳定重组和mRNA水平的表达 ,TGF β1 蛋白活性检测及流式细胞仪免疫荧光测定结果 ,均证实了克隆细胞表达并分泌TGF β1 蛋白。结论 :通过基因转移方法成功地在NIH/3T3细胞中重组并表达人TGF β1 蛋白 ,PCR结果还提示NIH/3T3细胞系基因组中无人TGF β1 序列存在。
Objective: To investigate the expression of and identify human TGF β1 in NIH/3T3 cells. Methods: Human recombinant TGF β1 was transfected into NIH 3T3 cells with the karyocyte expressive vector. The clone cells were selected by G418. All the clone cells were tested with southern blot, northern blot, PCR and RT PCR and a subclone of mink long cell line (CCL 64) was used to measure TGF β1 activity. Results: Human TGF β1 gene was stably recombined into NIH/3T3 cells and its mRNA was expressed in them. It was proved by flow cytometry and CCL 64 cell growth inhibition that clone cells secreted TGF β1 protein. Conclusion: Human TGF β1 is recombined and expressed in NIH/3T3 cells after the gene transfection. There is no human TGF β1 sequence in wild type NIH/3T3 cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2000年第1期68-71,共4页
Journal of Third Military Medical University
