用MAPs制备新肿瘤相关基因1 A6/DRIM的多克隆抗体

Preparation of a polyclonal antibody against N-terminal of 1A6/DRIM with MAPs method

目的 :获得 1A6 /DRIM的N端特异性抗体并对其在细胞内的定位进行分析。方法 :针对 1A6 /DRIM的N端多肽用多通道同步固相全自动合成系统合成 ,以无免疫原性的赖氨酸结构为核心偶联 ,将此多聚抗原肽 (mutipleantigenicpeptides,MAPs)免疫新西兰大白兔 ,从免疫兔血清中获得 1A6 /DRIM的多克隆抗体。结果 :用WesternBlot技术证明了 1A6 /DRIM的N端抗体 ,与C端特异性单克隆抗体识别相同的 1A6 /DRIM基因表达的 31 0 0 0 0蛋白条带 ,经过WesternBlot和免疫荧光的方法初步证实 1A6 /DRIM在多种胃癌细胞系、乳腺癌细胞系以及肝癌细胞系中表达 ,免疫荧光显示 1A6 /DRIM主要定位在细胞核内。结论 :对于用谷胱苷肽巯基转移酶 (Glutathion S transferase ,GST)融合蛋白方法不能诱导表达的蛋白 ,可利用合成MAPs制备抗原的方法获得抗体。本研究用此方法制备的抗体特异性识别 1A6

Objective: To obtain the antibody against N terminal of 1A6/DRIM, and thereafter get the profile of 1A6/DRIM expression in different cell lines. Methods: The N terminal of 1A6/DRIM (aa 577 714) was cloned into pGEX 4T 3. Multiple antigenic peptides (MAPs)(aa638 661) was synthesized as the antigen with Fmoc/PyBOP method. Rabbits were immunized by injecting the MAPs and the immunized sera were analyzed with ELISA and Western Blot. The Western Blot and immunofluorescence were performed to analyze the expressing profiles of the 1A6/DRIM in different tumor cell lines. Results: The antibody specifically recognized the full length of 1A6/DRIM as a 310 kDa band, which was also recognized by C terminal monoclonal antibody shown by Western Blot. 1A6/DRIM is expressed in multiple tumor cell lines and mainly located in the nuclei. Conclusion: Preparation of the antibody with MAPs is a useful technique when the fusion proteins can not be induced in E coli . The antibody we got via MAPs has supplied a good tool for further studies on the functions of the novel gene 1A6/DRIM.