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目的原核表达、纯化人细胞外基质磷酸糖蛋白(Mepe)基因,并制备多克隆抗体。方法设计出扩增人Mepe全长基因的特异引物,通过PCR扩增出该基因片段,测序正确后插入到含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克隆抗体,用ELISA测定抗体的效价,Western印迹鉴定抗体的特异性。结果原核表达了人Mepe全长基因,纯化了MEPE蛋白,并获得了抗MEPE的多克隆抗体,抗体效价达到1∶25 600,Western印迹结果显示,该抗血清能够特异识别原核及真核细胞表达的MEPE蛋白。结论 MEPE蛋白能够诱导小鼠产生具有较高效价和特异性的多克隆抗体,为进一步研究MEPE的功能奠定了基础。
Objective To purify human extracellular matrix phospholipoprotein (Mepe) gene by prokaryotic expression and prepare polyclonal antibody. Methods Specific primers for amplifying the full-length gene of human Mepe were designed. The gene fragment was amplified by PCR and inserted into prokaryotic expression vector pGEX-KG containing GST gene after sequencing correctly. The recombinant plasmid was induced by IPTG and transformed into glutathione Peptide agarose beads were used to purify the fusion protein. The purified protein was used to immunize mice to prepare polyclonal antibody. The antibody titer was determined by ELISA. The specificity of the antibody was identified by Western blotting. Results The full-length human Mepe gene was expressed in prokaryotic cells, the MEPE protein was purified and the anti-MEPE polyclonal antibody was obtained. The antibody titer reached 1:25 600. Western blotting showed that the antiserum specifically recognizes prokaryotic and eukaryotic cells Expressed MEPE protein. Conclusion MEPE protein can induce the production of polyclonal antibody with high titer and specificity in mice, which lays the foundation for further study on the function of MEPE.