人Snapin基因片段的原核表达及多克隆抗体的制备  

Prokaryotic expression and preparation of rabbit polyclonal antibody against human Snapin protein

作　　者：张沙沙[1] 陈中[1] 赵钟钟[1] 孙晓莉[1] 袁秀杰[1] 张朝[1] 

机构地区：[1]南京师范大学生命科学学院江苏省分子医学生物技术重点实验室,210046

出　　处：《免疫学杂志》2010年第8期710-713,共4页Immunological Journal

基　　金：国家自然科学基金资助项目(30570662;30871228);科技部重大科学研究计划资助项目(2006CB943503)

摘　　要：目的制备兔抗人Snapin的多克隆抗体,检测其特异性以及Snapin蛋白在心脏组织中的分布。方法构建pGEX-4T-1-Snapin N/C质粒,通过原核表达和纯化分别获得带有snapin基因N端(1-54Aa)和C端(60-136Aa)表达产物(融合蛋白GST-Snapin N/C),通过多点皮下注射免疫新西兰家兔,多次免疫后获得血清,用Protein G Agrose亲和纯化血清,获得IgG型高效价抗Snapin多克隆抗体。结果经过ELISA检测发现二种抗体效价高,滴度在1:256000时仍有较强信号。用Western blotting以及免疫组织化学检测发现二种抗体的特异性良好,能特异性识别小鼠心脏组织Snapin蛋白,其中尤以抗Snapin C抗体为佳。结论成功制备出高效价、强特异性的抗Snapin N/C抗体,为进一步研究Snapin蛋白在心脏中的作用提供了条件。This study aimed to construct prokaryotic expression vector for C and N terminus of Snapin and prepare high titer and specific antiserum of Snapin.We constructed pGEX-4T-1-Snapin N/C vectors in DH5α,and expressed the vector in E.coli BL21.Then we obtained GST fusion proteins that respectively recognize N and C terminus of human Snapin,which were subsequently purified and used to immunize female rabbits to get antiserum.ELASA assay indicated the titter of the antiserum to purified protein was 1：256 000,while analysis of Western blotting and immunohistochemistry showed that native cardiac snapin is specifically recognized by either anti-Snapin C and anti-Snapin N,especially anti-Snapin C.In this study,we successfully prepared GST fusion protein （GST-Snapin） and anti-human Snapin antibody with high titer and specificity,which provides a useful tool to investigate the expression and distribution of snapin in the heart and its functional role in cardiac E-C coupling.

关 键 词：Snapin 多克隆抗体 心脏 

分 类 号：R392.11[医药卫生—免疫学]