人硫氧还蛋白1多克隆抗体制备及对内毒素血症新生大鼠的保护作用

Preparation of polyclonal antibody of recombinant human thioredoxin-1 and its protective effects on neonatal rats with endotoxemia

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。

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: DNA 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.