重组人肝细胞生长因子的纯化及鉴定

Purification and identification of human recombinant hepatocyte growth factor expressed by CHO cells

目的对重组人肝细胞生长因子（Ｒｅｃｏｍｂｉｎａｎｔ ｈｕｍａｎ ｈｅｐａｔｏｃｙｔｅ ｇｒｏｗｔｈ ｆａｃｏｒ， ｒｈＨＧＦ）进行初步纯化及鉴定。方法 培养上清液经过离心、超滤，在ＦＰＬＣ系统上经肝素亲和层析分离纯化，用ＥＬＩＳＡ法检测、收集ｒｈＨＧＦ。ＳＤＳ－ＰＡＧＥ电 泳鉴定纯度和相对分子质量，大鼠原代肝细胞培养法检测ｒｈＨＧＦ的生物学活性。结果ｒｈＨＧＦ主要在０．９～１．３ ｍｏｌ／Ｌ ＮａＣｌ 范围内被洗脱，由约６２ ０００和３４ ０００两个亚基组成，明显促进大鼠肝细胞ＤＮＡ合成。结论 经肝素亲和层析分离，从 表达山ＨＧＦ的ＣＨＯ细胞培养上清液中纯化出有活性的ｒｈＨＧＦ。

Objective: To purify. and identify recombinant human hepatocyte growth facor (rhHGF) from the culture supernatant of CHO cells. Methods Affinity chromatography employing Heparin-Sepharose CL-6B column was performed to fractionate culture supernatant of CHO cells, and the fractions containing rhHGF were screened by ELISA. SDS-PAGE gel electrophoresis and the measurement of 3H-thymidine incorporation into primary culturerat hepatocytes were employed to identify rhHGF. Results The rhHGF was eluted when the concentration of NaCl was between 0.9 and l.3 mol/L. Purified rhHGF, which presented 2 bands on reduced SDS-PAGE (at 62 000 and 34 000, respectively), increased the 3H-thymidine incorporation into primary cultured rat hepatocytes. Conclusion Bioactive rhHGF can be purified by heparin affinity chromatography from the culture supernatant of CHO cells expressing rhHGF.