【摘要】 目的克隆人硫氧还蛋白1(hTrx-1)基因并在大肠杆菌表达体系中进行有效表达,制备其多克隆抗体。初步探讨hTrx-1对内毒素血症新生Sprague-Dawley(SD)大鼠的保护作用。方法从人胎肝细胞中用RT-PCR的方法得到了hTrx-1全长基因,将它克隆到原核表达载体上,在大肠杆菌诱导表达并纯化,纯化的蛋白免疫SD大鼠,制备多克隆抗体。将新生SD大鼠分为正常组、脂多糖(LPS)组和hTrx-1组(n=12)。正常组、LPS组分别予以腹腔注射生理盐水、LPS(5 mg/kg);hTrx-1组于LPS注射前30 min腹腔注射hTrx-1(10 mg/kg)。观察正常组、LPS组与hTrx-1组新生大鼠24 h死亡率。结果成功构建PET-28a-hTrx-1原核表达载体,表达纯化了hTrx-1,获得高效价多克隆抗体。正常组、LPS组与hTrx-1组大鼠24 h的死亡率分别是0、67%、17%(χ2=14.400,P<0.01)。结论成功制备了hTrx-1多克隆抗体;初步证明了该蛋白对内毒素血症新生SD大鼠具有保护作用。更多还原

【Abstract】 Objective To clone the gene human thioredoxin 1(hTrx-1) expressing its protein in the E.coli expression system and to obtain its polyclonal antibody,and to study the protective effects of hTrx-1 on neonatal rats with endotoxemia.Methods cDNA encoding hTrx-1 from fetal liver cells was isolated by RT-PCR.The hTrx-1 was cloned to the prokaryotic expression plasmid PET-28a to induce its protein expression in the E.coli expression system.The purified hTrx-1 was injected into rats to prepare polyclonal antibody.Newborn Sprague-Dawley rats were randomly assigned to three groups: control,lipopolysaccharide(LPS) and hTrx-1(n=12 each).The control and the LPS groups were intraperitoneally injected with normal saline and LPS(5 mg/kg),respectively.The hTrx-1 group received an intraperitoneal injection of hTrx-1(10 mg/kg) 30 minutes before LPS injection.The mortality rate 24 hrs after injection was compared between the three groups.Results The prokaryotic expression plasmid PET-28a-hTrx-1 was constructed.The hTrx-1 protein was expressed and purified.The polyclonal antibody of hTrx-1 with the titer of 1∶51200 was prepared.The mortality rate of the control,LPS and hTrx-1 groups was 0,67% and 17%,respectively(χ2=14.400,P<0.01).Conclusions The polyclonal antibody of hTrx-1 is prepared successfully.The hTrx-1 protein has protective effects on neonatal rats with endotoxiamia.更多还原