摘要
目的在CHO细胞中表达重组人促卵泡激素(Follicle-stimulating hormone,FSH)。方法用内部核糖体进入位点序列(IRES)将FSHα和β亚基在基因水平上进行拼接,并在同一启动子的控制下构建重组真核表达质粒pcDNA5/dhfr/FSH,转染CHO细胞,筛选并建立稳定表达工程细胞株,并对工程细胞的生长和表达进行分析。结果重组真核表达质粒pcDNA5/dhfr/FSH经双酶切和测序证明构建正确;转染CHO细胞获得高的阳性克隆率(80.65%),经MTX加压和克隆筛选获得稳定表达工程细胞株D5;D5细胞株在无血清摇瓶批次培养中FSH的表达量为2.5 IU/ml,在培养参数可控的生物反应器中FSH的表达量为摇瓶中的2倍多,达6 IU/ml。结论建立了一种在CHO细胞中表达异二聚体糖蛋白的方法,获得的工程细胞适应无血清培养且易于扩大培养,在生物反应器中可获得高的表达量,且具有进一步优化提高表达量的潜力。
Objective To express recombinant human follicle-stimulating hormone（FSH） in CHO cells.Methods Recombinant plasmid pcDNA5 / dhfr / FSH containing the α and β subunits of FSH linked at gene level with internal ribosome entry site（IRES） sequence was constructed under the control of a single CMV promoter and transfected to CHO cells,based on which a recombinant CHO cell strain for stable expression was screened and analyzed for growth and expression of target protein.Results Restriction analysis and sequencing proved that recombinant plasmid pcDNA5 / dhfr / FSH was constructed correctly,with which the positive cloning rate of transfected CHO cells was 80.65%.A recombinant CHO cell strain D5 was obtained by MTX pressure and cloning screening.The expression level of FSH in D5 cell strain in serum-free fed-batch culture in shake-flask was 2.5 IU / ml,which increased by more than 2 times and reached 6 IU / ml in bioreactor with controllable culture parameters.Conclusion A method for expression of heterodimeric glycoprotein in CHO cells was developed.The obtained recombinant CHO cells were suitable for serum-free and large-scale cultures,with which the target protein was highly expressed in bioreactor,and the expression level might be further increased by optimization of culture condition.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第12期1409-1412,共4页
Chinese Journal of Biologicals
关键词
促卵泡激素
异二聚体糖蛋白
CHO细胞
培养基
无血清
Follicle-stimulating hormone（FSH）
Heterodimeric glycoprotein
CHO cells
Medium
serum-free
