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目的:表达及纯化重组人分化抑制因子3(Id3)蛋白,制备兔抗人Id3多克隆抗体。方法:在大肠杆菌BL21(DE3)中表达并纯化重组人Id3融合蛋白,以纯化Id3重组蛋白为免疫原,免疫新西兰家兔,制备兔抗人Id3多克隆抗体,通过亲合层析实验去除抗血清中非特异性成分,免疫双向扩散、间接ELISA及Western blot法检测抗体效价和特异性。用间接免疫荧光试验(IFA)对人乳腺上皮癌细胞(MCF7)、人前列腺癌细胞(PC-3M)、人肺腺癌细胞(A549)细胞内Id3表达进行定位研究。结果:SDS-PAGE电泳、Western blot分析证实,表达载体Id3/pET-32a在大肠杆菌BL21中经诱导可大量表达His-Tag Id3融合蛋白;表达产物通过镍离子亲和层析法纯化得到相对分子质量(Mr)为34 000的Id3融合蛋白;Id3重组蛋白免疫家兔获得的抗血清效价分别为1∶8(双向扩散)和1∶8 000(ELISA),Western blot分析表明抗体具有抗人Id3的良好特异性。IFA结果发现,A549细胞内Id3表达量很低;在MCF7和PC-3M细胞内,Id3呈高水平表达,且主要定位于核浆。结论:重组人Id3蛋白的表达纯化及抗人Id3多克隆抗体的研制为进一步研究该蛋白的功能及其临床应用研究提供了有利工具。
OBJECTIVE: To express and purify recombinant human differentiation inhibitor 3 (Id3) protein and prepare a polyclonal anti-human Id3 antibody. METHODS: Recombinant human Id3 fusion protein was expressed and purified in E. coli BL21 (DE3). The purified recombinant Id3 protein was used as an immunogen to immunize New Zealand rabbits to prepare polyclonal antibody against rabbit polyclonal antibody against Id3. Anti-Id3 antibody was removed by affinity chromatography Antibody titer and specificity were detected by non-specific serum components, two-way immunoprecipitation, indirect ELISA and Western blot. The expression of Id3 in human breast epithelial carcinoma (MCF7), human prostate cancer (PC-3M) and human lung adenocarcinoma A549 cells was detected by indirect immunofluorescence assay (IFA). Results: SDS-PAGE electrophoresis and Western blot analysis confirmed that the expression vector Id3 / pET-32a was induced in E. coli BL21 to express a large amount of His-Tag Id3 fusion protein. The expressed product was purified by nickel ion affinity chromatography to obtain the relative molecule The Id3 fusion protein with a Mr of 34 000 was obtained. The titer of the antiserum obtained from the immunized rabbits with Id3 protein was 1: 8 (bidirectional diffusion) and 1: 8000 (ELISA), respectively. Western blot analysis showed that the antibody possessed anti-human Good specificity of Id3. IFA results showed that Id3 expression was low in A549 cells. Id3 was highly expressed in MCF7 and PC-3M cells, and mainly located in nucleus pulposus. Conclusion: The expression and purification of recombinant human Id3 protein and development of polyclonal anti-human Id3 antibody provide a useful tool for further study on the function and clinical application of this protein.