人肌红蛋白基因的克隆、表达及其抗体制备

Gene Cloning and Expression of Human Myoglobin and Preparation of Its Polyclonal Antibody

目的克隆、表达人肌红蛋白基因,并制备人肌红蛋白多克隆抗体。方法应用RT-PCR方法从人骨骼肌总RNA中扩增肌红蛋白基因(hMb)编码序列,克隆入原核表达载体pRSETc中,并在大肠杆菌E.coliBL21(DE3)中诱导表达。表达产物经亲和层析和凝胶层析纯化。纯化蛋白免疫小鼠,制备多克隆抗体,并用Westernblot检测其特异性。结果测序表明RT-PCR所得的肌红蛋白基因hMb序列与GenBank(NM203377)中报道的序列一致。该基因在大肠杆菌E.coliBL21(DE3)中获得高效可溶性表达,表达产物经纯化,获得电泳纯的蛋白。重组人肌红蛋白的表达水平达12.8%。每升发酵液中可获得纯化的目的蛋白6.438mg。Western blot分析表明该蛋白为融合有6个His的hMb蛋白。ELISA法测得抗体效价为1∶12800,Western blot证实其具有较好的特异性。结论已成功克隆人肌红蛋白基因,在大肠杆菌中获得了可溶性表达,并制备了抗人肌红蛋白的多克隆抗体。

Objective To express human myoglobin and prepare its polyclonal antibody. Methods The gene sequence encoding human myoglobin（hMb） from the total RNA of human skeletal muscle by RT-PCR and clone into prokaryotic expression vector pRSETc for expression in E. coil BL21 （ DE3 ）. Purify the expressed product by affinity chromatography and gel filtration chromatogra- phy. Imm,mize mice with the purified protein to prepare polyclonal antibody. Test the prepared antibody for specificity by Western blot.Results The sequence of amplified hMb gene was consistent with that reported in C, enBank （ NM203377 ）. The gene was highly expressed in a soluble form in E. coil BL21 （ DE3 ） , and the expressed product was electrophoreticaUy pure and contained 12. 8% of total somatic protein. Western blot proved the expressed product as hMb protein. A portion of 6. 438 mg of target protein was purified from 1 L of fermentation liquid. The polyclonal antibody induced in mice, with an ELISA titer of 1：12 800, showed good specificity as proved by Western blot. Conclusion Human myoglobin gene was successfully cloned and expressed in a soluble form in E. coli, and the polyclonal antibody against hMb was successfully prepared.