牙鲆甲状腺激素受体TRaA的原核表达和多克隆抗体的制备及应用

Prokaryotic expression and polyclonal antibody preparation of TRaA in Japanese flounder Paralichthys olivaceus

摘要:

为研究牙鲆甲状腺激素受体TRaA在牙鲆变态发育过程中的调控作用, 将TRaA基因克隆插入融合表达载体pET30a, 并在大肠杆菌Escherichia coli DE3(BL21)中进行诱导表达。表达菌株经1 mmol/L 异丙基-b-D-硫代半乳糖苷(IPTG)诱导4 h后, 重组蛋白TRaA表达并形成包涵体。SDS-PAGE和Western blotting检测鉴定表达产物。包涵体经变性后在His-Bind树脂进行亲和层析纯化, 柱上复性法对重组蛋白复性, 获得纯度较高的目的蛋白, 蛋白复性的效果良好。用纯化后的目的蛋白免疫新西兰家兔制备多克隆抗体。Dot blotting检测抗体效价达1:200 000, 检测证明抗体特异性良好。此外, 通过染色质免疫沉淀技术鉴定了在活体细胞中多克隆抗体与TRaA的特异性结合, 表明了甲状腺激素通过其受体在体内参与碱性磷酸酶(ALP)基因的转录调控。

Abstract:

To study the role of the thyroid hormone receptor TRaA involved in the process of the metamorphic development of Japanese flounder, we firstly cloned the TRαA gene, then ligated into the fusion expression vector pET30a and expressed in Escherichia coli DE3 (BL21) host cells. After induced for 4 h with 1 mmol/L Isopropyl b-D-Thiogalactoside, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The recombinant protein was denatured and purified by His-Bind resin, then renatured through gradient washing on His-bind resin column. After that, polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein. Dot blotting analysis showed the antibody with the titer of 1:200 000 reacted specifically to the expressed recombinant protein. Furthermore, a chromatin immunoprecipitation assay was performed to identify the specific binding between the antibody and TRαA in living cells of Japanese flounder. The result showed that thyroid hormone was involved in the alkaline phosphatase (ALP) gene transcriptional regulation through TRaA in vivo.