人β-NGF真核表达载体的构建及其表达产物的生物活性

目的在CHO细胞中共表达二氢叶酸还原酶(DHFR)基因及人神经生长因子β亚基(-βhNGF)基因,并对表达产物进行鉴定和生物活性分析。方法克隆DHFR和-βhNGF基因,测序,酶切,连接到真核表达载体pBudCE4.1中,构建人NGF基因重组表达质粒pBudCE4.1/DHFR/-βhNGF,经电转染法导入二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞(CHO-DHFR-)中,经添加Zeocin抗生素和撤除H、T(次黄嘌呤、胸腺嘧啶脱氧核苷)双筛选,获得了DHFR+细胞克隆。经MTX加压筛选高表达-βhNGF的CHO细胞株。SDS-PAGE、ELISA和Western blot对表达产物进行鉴定,并利用鸡胚背根神经节法检测表达蛋白的生物活性。结果人神经生长因子在CHO细胞中得到较高表达,表达量达0.3μg/ml,且表达产物具有良好的生物活性。结论为大规模工业化生产重组人神经生长因子奠定了基础。

Construction of Eukaryotic Expression Vector of Human β-NGF and Activity of Expressed Product

Objective To achieve the coexpression of dihydrofolic acid reductase(DHFR) and human nerve growth factor β subunit(β-hNGF) genes in CHO cells.Methods Clone DHFR and β-hNGF genes,identify by sequencing,digest with restriction endonuclease and insert into eukaryotic expression vector pBudCE4.1.Transfect the constructed recombinant plasmid pBudCE4.1/DHFR/β-hNGF to DHFR defective CHO(CHO-DHFR~-) cells by electrotransfection and screen DHFR~+ cell clones by adding Zeocin antibiotic and deleting hypoxanthine and thymidine.Select the CHO cell strain for high expression of β-hNGF by MTX pressure screening.Identify the expressed product by SDS-PAGE,ELISA and Western blot,and analyze its biological activity by chick embryo dorsal root nervous ganglion culture method.Results The expression level of β-hNGF in CHO cells reached 0.3 μg/ml,and the expressed product showed good biological activity.Conclusion The study laid a foundation of industrial production of rhNGF.