人心肌红蛋白多肽单抗原表位基因的克隆、原核表达及抗体制备

Gene synthesis, prokaryotic expression and antibody preparation of human heart Myoglobin protein single epitope peptides

目的本研究以原核表达单抗原表位的人心肌红蛋白(MB)肽段,制备不同表位的多克隆抗体。方法利用软件分析人心肌红蛋白的抗原表位,获得4个肽段(片段1、2、3、4);PCR扩增其基因序列,连接到表达载体p ET-GST,转化至E.coli BL21进行诱导表达。结果 GST-Sepharose 4B亲和层析柱进行MB-GST融合蛋白纯化,获得Mr28 000左右的融合蛋白,质量浓度为15 mg/L。免疫新西兰大白兔获得高效价的多克隆抗体。结论 4种多肽抗体均具有较好的特异性。

This study designed to acquire single-epitope human heart myoglobin peptides by using prokaryotic expression.PyMOL was used to screen the antibody of different epitopes of human serum myoglobin,and4 peptides（fragments 1,2,3,4） were obtained.Their gene sequences were amplified by PCR,and then connected to the expression vector pET-GST for protein expression in E.coli BL21.The expressed protein（MB-GST fusion protein） was purified by GST-Sepharose 4B.Data showed the relative molecular mass of the fusion protein was about28 kD,and the protein expression level was 15 mg/L.Then the purified protein was used to immunize New Zealand white rabbits for high titer polyclonal antibody.Western blot results showed that the 4 peptides antibodies possess good specificity.