人细胞外基质磷酸糖蛋白MEPE的表达纯化及抗体制备

Expression and purification of Mepe and preparation of its polyclonal antibody

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 express and purify matrix extracellular phosphoglycoprotein（MEPE）/gene Mepe in Escherichia coli,and prepare the polyclonal antibody against MEPE.Methods The specific primers were used to amplify full length of human Mepe gene by PCR,then the gene was inserted into the vector pGEX-KG containing GST gene,GST-MEPE fusion protein was expressed in E.coli,and purified by using Glutathione Sepharose 4B.Then anti-MEPE antiserum was prepared in mice,the titer was determined by ELISA and the specificity was identified by Western blot.Results Mepe was expressed and purified,the polyclonal antibody against MEPE was prepared,the titer of this antibody was about 1∶25 600,and MEPE expressed in prokaryotic and eukaryotic cells could be recognized by this antiserum.Conclusions MEPE protein can induce the effective and specific polyclonal antibody in the mice,it is the basis to understand the function of MEPE.