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目的:构建小鼠白细胞介素-1α原核表达载体,表达并纯化IL-1α蛋白,制备兔抗鼠IL-1α多克隆抗体,并对抗体特性进行初步的鉴定。方法:利用RT-PCR技术,从BALB/c小鼠脾脏cDNA中扩增出IL-1α的全长基因,酶切后连接至pET32a(+)原核表达载体,重组载体测序正确后转化至BL21(DE3)大肠杆菌。利用蛋白质原核表达自动诱导方案成功表达重组蛋白。重组蛋白经电洗脱纯化后用以免疫新西兰大白兔,获得了抗小鼠IL-1α的多克隆抗体,ELISA检测抗体效价。Western blot和流式细胞术(FCM)检测抗血清的特异性。结果:成功构建了重组表达载体pET32a(+)-IL-1α,表达的重组蛋白纯化后免疫新西兰大白兔,得到的多克隆抗体ELISA显示抗体效价可达1∶25 600。Western blot和FCM分析该抗体能特异性结合IL-1α。结论:利用重组的IL-1α蛋白成功制备了高效价、高特异性的兔抗IL-1α抗体,为进一步研究IL-1α的生物学功能奠定了基础。
OBJECTIVE: To construct a prokaryotic expression vector for mouse interleukin-1α and to express and purify IL-1α protein to prepare rabbit anti-mouse IL-1α polyclonal antibody. The antibody characteristics were preliminarily identified. Methods: The full-length gene of IL-1α was amplified from the spleen cDNA of BALB / c mice by RT-PCR and ligated into pET32a (+) prokaryotic expression vector. The recombinant vector was sequenced and transformed into BL21 DE3) E. coli. The prokaryotic expression of protein prokaryotic expression program successfully expressed recombinant protein. Recombinant proteins were purified by electroelution to immunize New Zealand white rabbits to obtain anti-mouse IL-1α polyclonal antibody. The antibody titers were detected by ELISA. Antisera were detected by Western blot and flow cytometry (FCM). Results: The recombinant plasmid pET32a (+) - IL-1α was successfully constructed and purified. The purified recombinant protein was immunized into New Zealand white rabbits. The obtained polyclonal antibody showed a titer of 1:25 600. The antibody can specifically bind to IL-1α by Western blot and FCM. Conclusion: The high titer and high specificity of rabbit anti-IL-1α antibody was successfully prepared by recombinant IL-1α protein, which laid the foundation for further study on the biological function of IL-1α.