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目的:克隆人mag1-基因,在大肠杆菌中进行表达并制备其抗体。方法:利用PCR方法从含有mag1-基因全长序列的pcDNA3-mag1-质粒上扩增mag1-基因开放阅读框(ORF),将其插入表达载体pET-28 a(+)中,构建重组表达载体pET-28 a-mag1-,用IPTG诱导目的基因在E.coli中的表达,并通过W estern印迹方法对表达产物进行鉴定。表达产物经过初步纯化后作为抗原免疫兔制备多克隆抗体。结果:所获得的序列经测序分析与源序列一致;SDS-PAGE分析表明,经0.1 mmol/L IPTG 37℃诱导6 h后在E.coli中有大量的融合蛋白表达,相对分子质量约为18×103。W estern印迹分析提示,诱导表达产物能与H is单抗发生特异反应。ELISA结果表明获得了高效价的多克隆抗体。结论:获得了H is-mag1-融合蛋白在大肠杆菌中高效表达,并制备了其相应的多克隆抗体。
OBJECTIVE: To clone human mag1-gene, express it in E. coli and prepare its antibody. Methods: The open reading frame (ORF) of mag1 gene was amplified from pcDNA3-mag1 plasmid containing the full length of mag1 gene by PCR and inserted into the expression vector pET-28 a (+) to construct a recombinant expression vector pET-28 a-mag1-. The expression of the target gene in E. coli was induced by IPTG, and the expressed product was identified by Western blotting. After primary purification, the expressed product was used as antigen to immunize rabbit to prepare polyclonal antibody. Results: SDS-PAGE analysis showed that the fusion protein was expressed in E.coli after being induced by 0.1 mmol / L IPTG at 37 ℃ for 6 h, and the relative molecular mass was about 18 × 103. Western blot analysis suggested that the expressed product could specifically react with His mAb. ELISA results showed that high titer polyclonal antibodies were obtained. Conclusion: H is-mag1-fusion protein was successfully expressed in E. coli and its corresponding polyclonal antibody was prepared.