抗nestin多克隆抗体的制备  

Preparation of polyclonal antibody to nestin

作　　者：李军林[1] 韩骅[1] 兰莉[2] 杨曦[1] 李军锋[1] 王亚蓉[3] 祝仰庭[1] 

机构地区：[1]第四军医大学基础部医学遗传学与发育生物学教研室,陕西西安710033 [2]第四军医大学唐都医院儿科,陕西西安710038 [3]第四军医大学唐都医院放射科,陕西西安710038

出　　处：《第四军医大学学报》2005年第8期691-693,共3页Journal of the Fourth Military Medical University

基　　金：陕西省自然科学基金(2002011)

摘　　要：目的: 在大肠杆菌中融合表达编码小鼠nestin肽链C段的cDNA序列,制备特异性多克隆抗体;方法: 采用RT PCR的方法获得编码小鼠nestin肽链C段的cDNA序列,将测序正确的片段克隆入pET 32a,构建pET 32a nestin C表达载体.在大肠杆菌BL 21中IPTG诱导下表达6×Hisnestin C融合蛋白,纯化后免疫新西兰兔,制备兔抗鼠nes tin血清,以WesternBlotting和免疫组化方法分析其特异性.结果: 6×His nestin C融合蛋白免疫新西兰兔成功获得多克隆抗体,WesternBlotting和免疫组化结果显示制备的抗体具有较高的特异性.结论: 构建了pET 32a nestin C表达载体,并在大肠杆菌中获得高效表达.该多克隆抗体效价高、特异性好.AIM: To clone and express the C-fragment of cDNA encoding mouse nestin peptide and to obtain specific polyclonal antibody.METHODS:The C-fragment of cDNA encoding mouse nestin peptide was obtained with the method of RT-PCR.The correctly identified sequence was cloned into the prokaryotic expression vector pET-32a to construct pET-32a-nestin-C expression vector.6×His-nestin-C fusion protein was expressed in E.coli BL21 with IPTG induction.After purification,the fusion protein was used to immunize New Zealand rabbits to get the rabbit anti-nestin serum.The specificity of the ployclonal anti-nestin antibody was analyzed by Western Blotting and immunohistochemical methods.RESULTS: The ployclonal anti-nestin antibody was successfully obtained by immunizing New Zealand rabbits with 6×His-nestin-C fusion protein.The specificity of antibody was confirmed by Western Blotting and immunohistochemical methods.CONCLUSION:pET-32 a-nestin-C expression vector is constructed and highly expressed in BL-21.

关 键 词：nestin基因 融合表达 抗体制备 

分 类 号：Q785[生物学—分子生物学]