摘要
目的:切胶纯化重组人促甲状腺激素亚基(hTSHβ),免疫家兔制备多克隆抗体,为人TSH测定试剂盒的国产化奠定基础。方法:将含有重组人促甲状腺激素β亚基(hTSHβ)的大肠杆菌表达产物进行SDS-PAGE分离,切取相应分子量的蛋白条带,研磨回收后用于免疫新西兰大耳朵白兔,ELISA监测血清中抗hTSHβ抗体的效价,饱和硫酸铵沉淀及亲和层析自血清中纯化抗体。结果:切胶后获得了高纯度的重组GST-hTSHβ蛋白,所免疫家兔产生了针对hTSHβ的多克隆抗体,效价可达1:12000。结论:切胶纯化的重组hTSHβ蛋白可成功诱导兔产生高效价的抗hTSHβ多克隆抗体。
Objective：To prepare polyclonal antibody against human thyrotropin β subunit with recombinant human TSHβ,expressed in Escherichia coli and purified from the PAGE gel,as the immunogen.Methods：Raw E.coli expressions of rhTSHβ were subjected to SDS-PAGE,gel pieces containing proteins corresponding to the molecular weight of GST-rhTSHβ were excised and homogenated in liquid nitrogen.Extracted protein was dissolved in PBS solution and used as immunogen to immune rabbits after being verified as GST-fusioned protein by western blot.The titter of the anti-hTSHβ antiserum was monitered by ELISA.The antiserum was collected after the titter reach 1：12 000 and then IgG was purified by chromatography followed by ammonium sulfate precipitation.Results：Highly purified GST-rhTSHβ protein was obtained from the gel,which could successfully induce the generation of high-tittered anti-hTSHβ antibody in the rabbits.Conclusion：Above mentioned procedure applies us an option for preparing recombinant proteins as the immunogen and generating antibodies from the rabbit.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2010年第5期428-431,共4页
Chinese Journal of Immunology
基金
天津市“重中之重”学科建设专项基金资助项目(05YFGDSF02700)
关键词
人促甲状腺激素
免疫
人促甲状腺激素抗体
重组蛋白
Human thyrotropin
Immunization
Antibody of human thyrotropin
Recombination protein
