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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。
AIM: To investigate the protein expression of transforming growth factor β1 (TGF β1) in eukaryotic cell lines. METHODS: Human TGF β1 was transfected into NIH / 3T3 cells by eukaryotic plasmid expression vector, identified by G418, cloned and amplified by Southern blot, Northern blot, PCR and RT PCR. Growth inhibition of lung epithelial cells (CCL 64) assayed the secretion of TGF β1 protein by cloned cells. RESULTS: The recombinant recombinant human TGFβ1 gene was confirmed to be stable in the genome of NIH / 3T3 cells by RT-PCR and flow cytometry (FCM). The results confirmed that the cloned cells secreted TGFβ1 protein. CONCLUSION: Human TGFβ1 protein is successfully recombined and expressed in NIH / 3T3 cells by gene transfer. PCR results also suggest that there is no human TGFβ1 sequence in the NIH / 3T3 cell line.