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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。
Objective: To prepare polyclonal antibodies against recombinant interleukin-27 (IL-27) fusion protein and to identify its activity. Methods: Epstein-Barr virus-inducible 3 (EBI3) and mature p28 gene fragments were amplified from mouse spleen cells by reverse transcription polymerase chain reaction (PCR), and the two fragments were ligated using the Gly4Ser linker. The murine IL-27 single-stranded fusion gene fragment was cloned into p ET-32a (+) vector. The positive recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The expression of the target protein was identified by SDS-PAGE and Western blot. New Zealand white rabbits were immunized with the purified IL-27 fusion protein to prepare polyclonal antibodies to rabbit anti-mouse IL-27 fusion protein. Western blot and ELISA were used for specificity and titer analysis. Results: The IL-27 single-chain fusion gene was amplified from the total RNA of mouse spleen cells and sequenced according to the expected sequence. The recombinant protein was induced by IPTG in E. coli BL21 (DE3) and SDS-PAGE showed the relative molecular mass of the expressed fusion protein (molecular weight, Mr) about 67 000; Western blot results show that the fusion protein can react with anti-6 × His monoclonal antibody; Immunization of New Zealand white rabbits after antiserum, Western blot results show that the serum can be specific In combination with the IL-27 fusion protein, the titer of the polyclonal antibody can reach 1: 300 by ELISA. CONCLUSION: The mouse IL-27 fusion protein was successfully expressed in E. coli and a rabbit anti-mouse IL-27 polyclonal antibody with higher titer and specificity was prepared.