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目的:克隆并原核表达甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP-2)的EGF、SP功能区蛋白,制备其多克隆抗体,初步分析两个功能区蛋白的免疫原性。方法:从人胎肝组织提取总RNA,反转录得到cDNA作为模板分别扩增MASP-2的EGF、SP片段基因,再分别连接至pGEX-6P-2表达载体诱导表达GST融合蛋白;分离纯化融合蛋白后免疫适龄BALB/c小鼠制备多克隆抗体,Western blot法检测抗体特异性,间接ELISA检测抗体效价。结果:成功构建pGEX-6P-2-EGF和pGEX-6P-2-SP,并表达GST-EGF、GST-SP两种融合蛋白;制备出两种目的片段的多克隆抗体,特异性高,与其他蛋白无交叉反应,间接ELISA测定EGF抗体效价大于1∶32 000,SP抗体效价大于1∶40 000。结论:获得了MASP-2的EGF、SP两个功能区蛋白的多克隆抗体,特异性强,效价高。
OBJECTIVE: To clone and prokaryotic express the EGF and SP functional domain proteins of mannan-binding lectin-associated serine protease 2 (MASP-2) and prepare their polyclonal antibodies. The immunogenicity of the two functional domains proteins was analyzed. Methods: The total RNA was extracted from human fetal liver tissue and the cDNA of MASP-2 was amplified by reverse transcription. The EGFP and SP fragments of MASP-2 gene were amplified by PCR and ligated into pGEX-6P-2 expression vector to induce the expression of GST fusion protein. The fusion protein was used to immunize BALB / c mice to prepare polyclonal antibody. The antibody specificity was detected by Western blot and the antibody titer was detected by indirect ELISA. Results: The pGEX-6P-2-EGF and pGEX-6P-2-SP were successfully constructed and expressed both GST-EGF and GST-SP fusion proteins. Polyclonal antibodies against the two target fragments were prepared with high specificity No cross-reaction of other proteins, indirect ELISA measured EGF antibody titers greater than 1:32 000, SP antibody titers greater than 1:40 000. Conclusion: The polyclonal antibody against two functional domains of EGF and SP of MASP-2 was obtained, which showed high specificity and high titer.