分泌型重组人胰岛素样生长因子-II（IGF-II）在中国仓鼠卵巢细胞中的表达。

Expression of secreted recombinant human insulin-like growth factor-II (IGF-II) in Chinese hamster ovary cells.

作者信息

Bekkari H, Sekkat D, Straczek J, Hess K, Belleville-Nabet F, Nabet P

机构信息

Department of Biochemistry, Medicine School, University of Nancy, Vandoeuvre, France.

出版信息

J Biotechnol. 1994 Jul 29;36(1):75-83. doi: 10.1016/0168-1656(94)90025-6.

DOI:10.1016/0168-1656(94)90025-6

PMID:7765161

Abstract

Chinese hamster ovary (CHO-KI) cells were cotransfected with a plasmid pcDNAI containing the human preproinsulin-like growth factor II cDNA linked downstream to the human cytomegalovirus promoter and with a plasmid containing the neomycin resistance gene (pMAM-neo). CHO neo+ were selected by growth in medium supplemented with G418 geneticin. After amplification, the neomycin-resistant clones were screened for IGF-II production. IGF-II produced was identified by dot blot and quantified by ELISA. The clones C24, C40 and C94 secreted IGF-II at about 350-400 ng per 10(6) cells per day. DNA analysis of C24 and C40 CHO cells by PCR demonstrated the presence of the IGF-II construct in the transfected cells, presumably integrated into the chromosomal DNA. IGF-II produced by CHO cells and purified by RP-HPLC was a mitogen for MCF-7 stimulating mitosis 2-fold.

摘要

将含有与人类巨细胞病毒启动子下游相连的人胰岛素样生长因子II前胰岛素原cDNA的质粒pcDNAI与含有新霉素抗性基因的质粒（pMAM-neo）共转染中国仓鼠卵巢（CHO-K1）细胞。通过在添加了G418遗传霉素的培养基中生长来筛选CHO neo+。扩增后，筛选新霉素抗性克隆的胰岛素样生长因子-II产生情况。通过斑点印迹鉴定产生的胰岛素样生长因子-II，并通过酶联免疫吸附测定进行定量。克隆C24、C40和C94每天每10⁶个细胞分泌约350 - 400 ng的胰岛素样生长因子-II。通过聚合酶链反应对C24和C40 CHO细胞进行DNA分析，证明转染细胞中存在胰岛素样生长因子-II构建体，推测其已整合到染色体DNA中。CHO细胞产生并通过反相高效液相色谱法纯化的胰岛素样生长因子-II是MCF-7的促有丝分裂原，可刺激有丝分裂增加2倍。