摘要
目的 制备高效价的 NDR2抗体 .方法 构建p Rset- A- ndr2 ,p GEX- 4 T- 1- ndr2 - a和 p GEX- 4 T- 1- ndr2 - b三种重组表达质粒 ,并分别在大肠杆菌中诱导表达相应的融合蛋白 ;用全长 GST- NDR2蛋白免疫兔 ,然后用 GST- NDR2 - A和 GST- NDR2 - B片段加强免疫 ;对包涵体形式表达的 6 His-NDR2进行初步的纯化 ;利用固定于硝酸纤维素膜上的 NDR2抗原亲和吸附纯化抗血清 .结果 经免疫得到了高效价的兔抗人 NDR2多克隆抗血清 ,亲和纯化提高了 NDR2抗体的特异性 ;免疫组化表明 NDR2是一种胞浆蛋白 .结论 用全长NDR2蛋白免疫兔 ,再用片段加强免疫 ,可以提高蛋白的抗原性 ,制备得到高效价的抗体 .纯化后的特异性 NDR2抗体 ,为进一步研究
AIM To obtain an anti NDR2 polyclonal anti body. METHODS Full length of ndr 2 cDNA was cloned into pRset A vector and two truncated sequences of ndr 2 were cloned into pGEX 4T 1 vector respectively. The fusion proteins were expressed in E.coli and partially purified by inclusion body fraction. Immunization in rabbits with the whole NDR2 protein was reinforced with two truncated fragments of NDR2 twice. The anti NDR2 specific antibody was purified by absorbing antiserum with NDR2 antigen immobilized NC filter. RESULTS High titer of antiserum against NDR2 was obtained in immunized rabbits and the specificity to NDR2 was increased after affinity purification. Immunohistochemical test with this antibody showed that NDR2 is a cytoplasmic protein. CONCLUSION Reinforcement with truncated NDR2 proteins can strengthen the antigenicity of NDR2 protein in rabbits. The specific NDR2 antibody can be obtained by NC filter affinity purification.
出处
《第四军医大学学报》
北大核心
2002年第8期716-720,共5页
Journal of the Fourth Military Medical University
基金
国家自然科学基金资助项目 (3 0 0 70 773 )
