人碱性成纤维细胞生长因子基因在COS-7细胞中的表达及活性检测  

THE EXPRESSION AND ACTIVITY OF NEWLY-CLONED HUMAN BASLC FIBROBLAST GROWTH FACTOR GENE IN COS-7 CELL

作　　者：于凤山[1] 段德义[1] 陈立南[1] 秦丽华[1] 徐群渊[1] 姜传涛[1] 

机构地区：[1]首都医科大学北京神经科学研究所,北京100054

出　　处：《解剖学报》2000年第z1期76-80,共4页Acta Anatomica Sinica

摘　　要：目的 获得人碱性成纤维细胞生长因子（ｂＦＧＦ），用于治疗神经系统疾病；克隆添加 Ｋｏｚａｋ序列的 ｂＦＧＦ ｃＤＮＡ序列，用于真核细胞表达。方法 提取国人脑胶质瘤细胞系 ＢＴ３２５总 ＲＮＡ，ＲＴ－ＰＣＲ法扩增 ｂＦＧＦ ＣＤＮＡ片段，重组于 ｐＧＥＭ－３ｚｆ（＋）载体，测序正确后构建表达载体，转染猴肾成纤维细胞系 ＣＯＳ－７，Ｗｅｓｔｅｒｎ Ｂｌｏｔ检测表达，收集培养上清用 ＰＣ－１２进行活性检测。结果ＲＴ－ＰＣＲ扩增到４７３ｂｐ的带有Ｋｏｚａｋ序列的ｃＤＮＡ片段；成功构建了真核表达载体；在ＣＯＳ－７得到了表达，并有一定的生物活性。结论 由克隆到的ｂＦＧＦ　ｃＤＮＡ片段构建的真核表达载体能在ＣＯＳ－７中表达、分泌．并具有生物活性。Objective　The cDNA fragment of human basic fibr oblast growth factor (bFGF) with additional Kozak sequence was cloned and was th en expressed in eukaryotic cells in order to investigate the roles of bFGF in tr eatment of diseases in the nervous system. Methods T otal cellular RNA of glioma BT325 was isolated and RT-PCR amplification of the c DNA fragments was performed. The fragment was cloned into pGEM-3zf(+) vector an d sequenced. The eukaryotic expression vector was recombined and transfected into fibroblast cell line COS-7. bFGF expression was analysized using Western blot. The effect of bFGF on cultured PC-12 was observed. Results The DNA fragment of 473bp with additional Kozak sequence was amplified from reverse transcription mixture. Eukaryotic expression vector pCI-bFGF was r e combined and expressed in COS-7. The biological activity was shown on the cultu red PC-12 cell.Conclusion Full-length of cDNA of hu man bFGF with additional Kozak sequence was cloned and expressed in COS-7. This expressed product showed biological activity on certain cultured cell.

关 键 词：碱性成纤维细胞生长因子(bFGF) RT-PCR CDNA克隆 Western BLOT 

分 类 号：R322[医药卫生—人体解剖和组织胚胎学]