人Ⅱ型成对免疫球蛋白样受体β基因(PILRβ)的克隆、表达、纯化及其多克隆抗体的制备

Cloning and purification of paired immunoglobin-like type 2 receptor beta and preparation of its polyclonal antibody

目的:表达和纯化人Ⅱ型成对免疫球蛋白样受体蛋白PILRβ,制备并鉴定其多克隆抗体。方法:应用RT-PCR从人的外周血细胞总RNA中反转录并扩增出PILRβ基因,将其克隆到表达载体pET-32a,在大肠杆菌中IPTG诱导表达,经Ni2+-NTA亲和层析柱纯化,纯化后的蛋白多次注射免疫新西兰大白兔,获取多克隆抗血清,ELISA法检测抗体效价,Western印迹检测抗体特异性。结果:获得了较高纯度的PILRβ蛋白(Mr约为42 000)及其抗体,多抗效价达到1:10 000,且特异性较好。结论:成功克隆了PILRβ基因并表达纯化了其蛋白,获得了效价较高、特异性较强的多克隆抗体,为进一步研究PILRβ蛋白的功能和潜在的药物开发打下了基础。

Objective：To express and purify paired immunoglobin-like type 2 receptor beta （PILRβ） and prepare the polyclonal antibody against its protein. Methods： The PILRβ cDNA was amplified from the total RNAs extracted from human peripheral blood cells by RT-PCR and was cloned into pET-32a vector. The vector harboring PILRβ gene was then expressed in E. coli by IPTG induction and the product was purified by Ni^2＋-NTA affinity chromatography. Polyclonal antibody was prepared by immunizing rabbits with the purified protein. The titer and specificity of the antibody were examined by ELISA and Western blot, respectively. Results： Highly purified PILRβ protein （Mr 42 000） was obtained. The prepared polyclonal antibody was highly specific and had a titer of 1 ： 10 000. Conclusion： PILRβ gene is successfully cloned and the purified PILRβ protein is expressed. Polyclonal antibody against PILRβ（with high titer and specificity） is successfully obtained, which provides a basis for further study on PILRβ.