重组小鼠白细胞介素-27融合蛋白多克隆抗体的制备及鉴定

发布时间：2018-04-10 21:23

本文选题：小鼠白细胞介素- + 重叠延伸PCR　；参考：《重庆医科大学学报》2017年02期

【摘要】：目的:制备重组小鼠白细胞介素-27(interleukin-27,IL-27)融合蛋白的多克隆抗体并鉴定其活性。方法:用反转录PCR从小鼠脾细胞扩增出EB病毒诱导基因3(EBI3)和p28成熟肽基因片段,采用重叠延伸PCR用(Gly4Ser)3接头将2个片段进行连接,将得到的小鼠IL-27单链融合基因片段克隆于p ET-32a(+)载体。阳性重组质粒转化大肠杆菌BL21(DE3),异丙基硫代-β-D-半乳糖苷(IPTG)诱导融合蛋白表达,利用SDS-PAGE与Western blot鉴定目的蛋白的表达。以纯化的IL-27融合蛋白免疫新西兰大白兔,制备兔抗小鼠IL-27融合蛋白的多克隆抗体,利用Western blot和ELISA进行特异性及效价分析。结果:从小鼠脾细胞总RNA中扩增得到IL-27单链融合基因经测序与预期序列一致;在大肠杆菌BL21(DE3)中经IPTG诱导,SDS-PAGE显示表达出的融合蛋白相对分子质量(molecular weight,Mr)约67 000;Western blot结果表明该融合蛋白能与抗6×His的单克隆抗体发生反应;该蛋白免疫新西兰大白兔后获得抗血清,Western blot结果显示该血清可以特异性地与IL-27融合蛋白结合,ELISA测定该多克隆抗体的效价可以达到1∶3 200。结论:在大肠杆菌中成功表达出小鼠IL-27融合蛋白,并制备出效价较高、特异性较好的兔抗小鼠IL-27多克隆抗体。
[Abstract]:Aim: to prepare the polyclonal antibody against recombinant mouse interleukin-27 interleukin-27 (IL-27) fusion protein and to identify its activity.Methods: Epstein-Barr virus inducible gene (EBI3) and p28 mature peptide gene fragments were amplified from mouse spleen cells by reverse transcription PCR. The two fragments were ligated by overlapping extension PCR using Gly4Serf3 junction.The single strand fusion gene fragment of mouse IL-27 was cloned into pET-32a () vector.Positive recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and isopropylthio- 尾 -Dgalactoside (IPTG) induced the expression of fusion protein. The expression of the target protein was identified by SDS-PAGE and Western blot.The polyclonal antibody of rabbit anti-mouse IL-27 fusion protein was prepared by immunizing New Zealand white rabbits with purified IL-27 fusion protein. The specificity and titer of Western blot and ELISA were analyzed.Results: the single strand IL-27 fusion gene was sequenced from the total RNA of mouse spleen cells, and the sequence was consistent with the expected sequence.IPTG induced SDS-PAGE showed that the fusion protein could react with monoclonal antibody against 6 脳 His. The relative molecular weight of the fusion protein was about 67000 blot. The results showed that the fusion protein could react with the monoclonal antibody (McAb) of 6 脳 His in E. coli BL21DDE3. The results showed that the fusion protein could react with the monoclonal antibody of 6 脳 His.After immunizing New Zealand white rabbits, the antiserum was obtained by Western blot. The results showed that the antibody titer of the polyclonal antibody could reach 1:3 200 by Elisa with IL-27 fusion protein binding protein.Conclusion: mouse IL-27 fusion protein was successfully expressed in Escherichia coli and rabbit polyclonal antibody against mouse IL-27 was prepared with higher titer and better specificity.
【作者单位】：潍坊医学院医学检验学系;潍坊医学院病原生物学教研室;
【基金】：国家自然科学基金资助项目(编号:81170080、81470001) 潍坊医学院2011年科技创新前沿探索重点研究资助项目(编号:K11TS1004)
【分类号】：R392

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