摘要
本实验引物延伸合成人红细胞生成素信号肽序列 ;信号肽序列和人可溶性B淋巴细胞刺激因子 (hu mansolubleBLymphocyteStimulator,hsBLyS)基因重叠延伸拼接成融合基因 ;融合基因插入pcDNA3、pcDNA3 1、pEFneo质粒 ;信号肽引物设计时 ,突变同义密码 ,最终重组质粒成为分泌表达质粒 ;磷酸钙共沉淀将表达质粒和标志质粒pSV dhfr,共转染CHO—dhfr- 细胞 ;选择培养液筛选 ,氨甲喋呤扩增表达 ,获稳定表达rhsBLyS细胞株 ;ELISA检测浓缩表达上清 ,结果表明 :rhsBLyS表达量由 0 13 μg/mL上升至 0 5 5 μg/mL .
The B Lymphocyte Stimulator(BLyS), a novel immune regulator, is the most recent addition to the tumor necrosis factor family(TNF) ligands. BlyS induces B cell proliferation, differentiation and immunoglobulin secretion, and involves in immune response of T cell. It also strongly suppresses the growth of tumor cell lines. Fourthermore, BLyS is associated with the development of some autoimmune disease. Here, EPO signal peptide sequence and hsBLyS gene were linked by SOE method. The fusion product was cloned into eukaryotic plasmids:pcDNA3?pcDNA3.1?pEFneo, respectively. Meanwhile, the EPO signal peptide sequence was mutated so as to form a restriction enzyme cut site:BlnⅠ. Thus the recombinant plasmid can be used as secreting plasmid expressing other gene. The recombinant expression plasmid and marker plasmid: pSV-dhfr were co-transfected into CHO-dhfr^-cells. After screened and amplified by selection medium and MTX respectively, we constructed a CHO cell line which secreting express rhsBLyS stably. The level of rhsBLyS expression raised form 0.13?μg/mL to 0.55?μg/mL under the pressure of MTX.
出处
《南京师大学报（自然科学版）》
CAS
CSCD
2004年第2期81-86,共6页
Journal of Nanjing Normal University(Natural Science Edition)
基金
国家自然科学基金资助项目 ( 3 0 2 70 193 ) .
