小鼠17β-hsd10的克隆、表达及双抗夹心ELISA方法的建立

Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17β-hsd10 in mouse

原核表达小鼠17β-羟基类固醇脱氢酶(17β-hsd10),制备抗17β-hsd10多克隆抗体,建立17β-hsd10双抗夹心酶联免疫检测方法 (Enzyme linked immunosorbent assay,ELISA)。采用RT-PCR技术克隆得到小鼠肝脏17β-hsd 10基因,构建原核表达体系p ET15b-17β-hsd10/Escherichia coli BL21(DE3),IPTG诱导重组蛋白表达,并优化表达条件;目的蛋白用His融合蛋白纯化柱(His-Binding-resin)纯化并免疫BALB/c小鼠和新西兰大耳白兔,免疫血清总Ig G采用硫酸铵沉淀法纯化,用获得的高效价鼠源和兔源两种多克隆抗体,建立17β-hsd10双抗夹心酶联免疫检测方法。表达蛋白约为29.5 k Da,纯化后浓度为1.5 mg/m L,鼠源和兔源多克隆抗体效价分别为1.25×104和2.5×104,建立的检测方法对于多种羟基类固醇脱氢酶无交叉反应,特异性良好,可检出含量约为0.05-0.1μg/m L的阳性血清。建立的17β-hsd10双抗夹心ELISA方法,简便、快速、敏感,适合实验室研究科研广泛应用。并可能为检测阿尔茨海默病提供新思路和方法。

We expressed 17β-hydroxysteroid dehydrogenase10（17β-hsd10） recombinant protein, prepared anti-17β-hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay（ELISA） test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system p ET15b-17β-hsd10/Escherichia coli BL21（DE3） which induced with isopropyl-1-thio-β-galactopyranoside（IPTG） for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total Ig Gs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/m L of 17β-hsd10 protein with molecular weight of 29.5 k Da, and polyclonal antibodies from mouse and rabbit with the tite 1.25×10^4 and 2.5×10^4respectively. The concentration of 0.1 μg/m L of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.