【摘要】 为建立稳定表达人可溶性B淋巴细胞刺激因子(hsBLyS)的中国仓鼠卵巢(CHO)细胞系,从人胎盘cDNA文库中扩增hsBLyS基因,将人红细胞生成素(EPO)信号肽序列和hsBLyS基因重叠延伸为融合基因;融合基因分别插入pcDNA3、pcDNA3.1、pEFneo质粒;磷酸钙共沉淀将表达质粒和标志质粒pSVdhfr,共转染CHOdhfr细胞;选择培养液筛选,经氨甲喋呤选择压力扩增表达,获稳定表达hsBLyS的细胞系,表达量由0.13μg/ml上升至0.55μg/ml;同时利用pEFneo/hsBLyS重组质粒免疫BALB/c小鼠,检测结果表明小鼠产生hsBLyS的特异性抗体。本实验建立了稳定表达hsBLyS的CHO细胞系,对其基因免疫小鼠抗体产生的特点做了初步探讨,为hsBLyS进一步研究奠定了良好的基础更多还原

【Abstract】 To construct a CHO cell line secretory expressing hsBLyS (Human soluble B Lymphocyte Stimulator), a cDNA fragment encoding hsBlyS gene was amplified by PCR from cDNA library of the human placenta. The hsBLyS cDNA fragment and EPO signal peptide sequence were linked by the SOE method. The fusion sequence was cloned into eukaryotic plasmids: pcDNA3, pcDNA3.1 and pEFneo, respectively. The recombinant expression plasmid and marker plasmid pSV-dhfr were co-transfected into CHO-dhfr- cells. After screening and amplifying with selection medium and MTX respectively, the hsBLyS expression level rose from 0.13 μg/ml to 0.55 μg/ml. To study the immune response in mice immunized with pEFneo/hsBLyS plasmid, the plasmid was injected intramuscularly into BALB/c mice. The result of ELISA and Western blot showed that the recombinant plasmid could elicit high specific antibodies更多还原