正常人脑胶质纤维酸性蛋白的提取及鉴定

PURIFICATION OF GLIAL FIBRILLARY ACIDIC PROTEIN FROM NORMAL HUMAN BRAIN

经匀浆反复沉淀,DEAE—Sephadex A50 柱层析等多步骤,从正常成人脑白质中提取了胶质纤维酸性蛋白(GFAP)。提取物与兔抗人GFAP抗血清存在有特异性沉淀反应,提取物纯度为91％,分子量为43kd。用提取物免疫动物制备抗血清,以此作为第一抗体进行免疫细胞化学染色,结果可显示出胶质瘤细胞内GFAP的分布,证明提取物确为人GFAP。GFAP的提取为进而解决其抗体来源问题打下了基础。

Glial fibrillary acidic protein (GFAP) from normal human brain was extracted and purified. The white mater was homogenized with pH 8.0, 0.05mol/L sodium phosphate buffer. Suspensions were centrifuged at 105 000g for 1 hour. Then, the purified preparations were obtained with 25% ammonium sulfale prepitation and pH 5.0 acid precipitation, followed by DEAE-Sephadex A50 column chromatography. SDS-polyacrylamide gel electrophoresis and immunodiffusion with GFAP antiserum were used to monitor each step of the purification procedure. Purity of the preparations was 91% as indicated by the relative dye binding on acrylamide gel scanned with laser densitometer. The molecular weight of the preparation was 43 kd, and it reacted specifically with GFAP antiserum.BALB/c mice were immunized with the preparation, and the antiserum reacted specifically with GFAP in the glial cells (JCBI) .