摘要
目的获取高纯度的重组人源性肝细胞生长因子(rhHDSSF),并探讨rhHDSSF在体外的生物学活性。方法收获大量培养的表达细胞,以冻融法提取蛋白质;以IMAC pH递减非变性法纯化表达的rhHDSSF;以MTT法测定其对HepG2,T24,Fibroblast和Hela细胞的增殖效应。结果从细胞裂解物中纯化得到20 kD左右的单一蛋白条带,其纯度达89％。细胞增殖实验证实这一蛋白产物对HepG2,Hela细胞有显著的增殖作用,并表现出良好的量效和时效关系,而对T24,Fibroblast细胞则增殖效果不明显。结论 本研究证实,人源性肝细胞生长因子(HDSSF)转染细胞株中有20 kD左右HDSSF相应蛋白表达,与HDSSF、598 bp开放读码框理论值相一致,且纯度达89％,并发现其有良好的细胞增殖作用。rhHDSSF是一种非特异性促细胞增长物质,但它也有一定的选择性,这可能与不同细胞表达的细胞受体差异而导致蛋白信号传导不同有关。
Objective To obtain the high purity recombinated human hepatocyte DNA synthetic stimulating factor (rhHDSSF), and detect its biological activity in vitro. Methods A large amount of expression cells were harvested, the protein of the expression cells was extracted with freeze-thawed method and rhHDSSF was purified by IMAC pH decreasing progressively no-denaturing methods, rhHDSSF proliferation effect on HepQ, T24, fibroblast and Hela cell was detected with MTT assay. Results 20 KD single protein strip was obtained from splitting cells after purification, its purity was up to 89%. Cell proliferation test confirmed that this protein product had remarkable proliferative effect on HepQ, Hela cells; but no obvious proliferative effect was observed on T24 and fibroblast cells. Conclusion The research confirmed that HDSSF transfecting cell had about 20 KD HDSSF 598 bp corresponding protein expression, and the purity was up to 89% . The protein product had gcod proliferative effect on the cells.
出处
《中国感染控制杂志》
CAS
2003年第2期86-88,共3页
Chinese Journal of Infection Control
