摘要
脑红蛋白 (NGB)是新发现的脑特异的携氧蛋白 .为了对其功能进行深入的研究 ,应用已构建大鼠NGB基因编码区原核表达载体pGEX 4T 2 NGB转化的大肠杆菌BL2 1 ,获得可溶性表达产物 .用谷胱甘肽S 转移酶 (GST)亲和层析柱纯化后作为抗原 ,免疫Balb c小鼠 .通过传统的细胞融合方法制备了抗大鼠NGB的单克隆抗体 ,并对全部 4株单抗进行了亚类和亚型、效价和特异性鉴定 .
Neuroglobin (NGB), a recently identified monomer globin in nerve system, is highly affinitive with oxygen similar to myoglobin. After the coding region of rat NGB gene was sub cloned into the prokaryotic expression vector pGEX 4T 2, the recombinant vector was introduced into E. coli BL21 (DE3) cells for efficient expression. The soluble GST fusion protein was purified through GST column and used to immunize Balb/c mouse. The monoclonal antibody was then produced through hybridoma cells prepared following traditional protocol. Finally, four kinds of cell line highly secreting anti NGB monoclonal antibody were successfully screened. The Ig subclass, subtype, titer and specificity of the monoclonal antibody were also identified. The monoclonal antibody of rat NGB will facilitate further functional study of NGB.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2003年第6期751-756,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金重大研究计划 (No .90 2 0 80 17)
国家海外青年学者合作研究基金 (No .3 0 12 80 10 )
国家自然科学基金面上项目 (No .3 0 10 0 0 49
No .3 990 0 0 41
No.3 990 0 0 74)
北京市自然科学基金 (No .70 0 2 0 3 0 )资助~~
关键词
大鼠
脑红蛋白
原核可溶性表达
单克隆抗体
rat, neuroglobin, prokaryotic soluble expression, monoclonal antibody, preparation and characterization
