抗人Id3蛋白多克隆抗体的制备及其在肿瘤细胞内的定位研究  

Preparation of rabbit antibody against human Id3 and identification of intracellular localization of Id3 in tumor cells

作　　者：李晓军[1] 仲爱芳[1] 王凯[1] 贾丽[1] 陈芳芳[1] 虞伟[1] 

机构地区：[1]南京军区南京总医院全军临床检验医学研究所,江苏南京210002

出　　处：《细胞与分子免疫学杂志》2010年第9期871-873,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：江苏省"科教兴卫"医学重点人才基金课题(RC2007117);南京军区医药卫生科研项目重点课题(07Z032)

摘　　要：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。AIM： To express and purify the recombinant human inhibitor of differentiation 3（Id3） in E.coli and prepare rabbit polyclonal antibody against Id3.METHODS： The expression vector Id3/pET32a was transformed into E.coli BL21（DE3） and expression of histidine（His）-tagged Id3 fusion protein was induced with IPTG and confirmed by SDS-PAGE and Western blot techniques.Id3 fusion protein was purified through immolbilized Ni2＋ absorption chromatographic column.The purified protein was used to immunize rabbits.The titer and specificity of rabbit antisera were evaluated by immunodouble gel diffusion,ELISA and Western blot techniques.The collected rabbit antisera were purified with polypeptide affinity column.The intracellular distributions of Id3 expression in human human breast cancer cells（MCF7）,prostate cancer cells（PC-3M） and lung adenocarcinoma cells（A549） were observed by indirect immunofluorescence assay（IFA）.RESULTS： Id3 fusion protein with 6×his-tag was successfully expressed in E.coli BL 21.The expressed Id3 with the relative molecular mass size of 34 kD was purified specifically through affinity chromatographic column and confirmed by Western blot.The titers of the rabbit anti-Id3 polyclonal antibodies were 1∶8 by immunodouble gel diffusion and 1∶8 000 by ELISA.Western blot revealed that the prepared antisera reacted specifically with Id3.IFA showed that in A549,MCF7 and PC-3M cells,Id3 was mainly distributed in nucleoplasm and the Id3 expression level was significantly higher in MCF7 and PC-3M than in A549 cells.CONCLUSION： The expression and purification of human Id3 protein and preparation of polyclonal antibody against Id3 provide useful tools for the further study of Id3.

关 键 词：分化抑制因子3(Id3) 抗体 A549细胞 

分 类 号：R392.12[医药卫生—免疫学]