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

摘要:

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pET15b-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IgG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 kDa，纯化后浓度为1.5 mg/mL，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 ?0.1 μg/mL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。

Abstract:

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 pET15b-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 IgGs 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/mL of 17β-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25×104 and 2.5×104 respectively. The concentration of 0.1 μg/mL 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.