摘要
目的将密码子优化的一系列长效促红细胞生成素(Erythropoietin,EPO)基因定点整合至CHO-K1细胞株中,获得高效稳定表达的细胞株。方法对EPO基因定点突变得到Darbepoetin-1序列(专利公开序列)及其同义密码子序列Darbe-poetin-2、Darbepoetin-3,并构建相应重组表达质粒pcDNA5/FRT/dbp(s),采用定点整合技术,转染CHO-K1细胞株,经ELISA和荧光定量PCR法鉴定转染细胞株,比较同一整合位点同义密码子Darbepoetin alfa蛋白表达水平的差异。蛋白样品经蓝胶亲和层析、凝胶过滤和离子交换层析后,进行SDS-PAGE、Western blot及等电聚焦电泳分析。结果重组表达质粒pcDNA5/FRT/dbp(s)经测序鉴定正确;ELISA检测表明,Darbepoetin-2的表达水平明显优于Darbepoetin-1和Darbepoetin-3,最高可达1 845 IU/ml;SDS-PAGE分析显示,纯化的Darbepoetin alfa比rHuEPO具有更高的相对分子质量,可与兔抗人EPO多抗特异性结合,且比rHuEPO具有更高的唾液酸丰度。结论获得了高效稳定表达重组Darbepoetin alfa的CHO-K1细胞株。
Objective To obtain the CHO-K1 cell strain for stable and high expression of recombinant long-acting erythropoietin（EPO） by codon optimization and site-specific integration technique.Methods Darbepoetin-1 sequence（the patent reported sequence） and its synonymous codon sequences Darbepoetin-2 and Darbepoetin-3 were obtained by site-directed mutation EPO gene,based on which the corresponding expression vectors pcDNA5 / FRT / dbp（s） were constructed and transfected to CHO-K1 cell strain by site-specific integration technique.The transfected cell strains were identified by ELISA and fluorescent quantitative PCR,and the expression levels of Darbepoetin alfa protein with synonymous codons at the same integration site were compared.The expressed protein was purified by blue-gel affinity chromatography,gel filtration chromatography（GFC） and ion exchange chromatography（IEC）,then analyzed by SDS-PAGE,Western blot and isoelectric focusing electrophoresis.Results Sequencing results proved that recombinant plasmids pcDNA5 / FRT / dbp（s） were constructed correctly.ELISA showed that the expression level of Darepoetin-2 was significantly higher than those of Darbepoetin-1 and Darbepoetin-3,and reached 1 845 IU / ml at most.SDS-PAGE proved that purified Darbepoetin alfa,with high relative molecular mass and sialic acid abundance as compared with those of rHuEPO,showed specific binding toe rabbit anti-human EPO polyclonal antibody.Conclusion The CHO-K1 cell strain for high and stable expression of recombinant Darbepoetin alfa.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第8期898-902,共5页
Chinese Journal of Biologicals
