Lin Huayuan, Chen Yanjun, Huang Qiqi, Guo Xiaoquan, Liu Ping, Liu Weilian, Zhang Caiying, Cao Huabin, Hu Guoliang
Clinical Veterinary Laboratory, College of Animal Science and Technology, Jiangxi Agricultural University, Jiangxi, China.
Clinical Veterinary Laboratory, College of Animal Science and Technology, Jiangxi Agricultural University, Jiangxi, China.
Int J Biol Macromol. 2016 Jun;87:341-7. doi: 10.1016/j.ijbiomac.2016.03.001. Epub 2016 Mar 4.
Xanthine oxidase (XOD) is the members of the molybdenum hydroxylase flavoprotein family and it plays a vital role in the body's purine catabolism. In this study, we cloned the XOD 37kDa subunit protein by using RT-PCR and pMD-18-T clone vector based on the total RNA extracted from chicken liver. The cloning XOD subunit protein gene was ligated into the pET-32a to construct the recombinant plasmid pET-XOD. After the pET-XOD expression vector was transformed into host cells Rosetta (DE3), the recombinant XOD subunit proteins (54.8kDa) were successfully induced by isopropy1 β-d-thiogalactoside (IPTG). Rabbit antiserums were produced by using the purification of the recombinant XOD subunit protein as antigen. The titer of the antiserum was more than 1:102,400 determined by using ELISA. The result of Western blot demonstrated that the antiserum could specifically recognize the chicken liver XOD. Immunohistochemistry and immunofluorescence showed that the XOD mainly presented in the cytoplasm of chicken hepatocytes and proximal tubular epithelial cells. Our results indicated that the XOD subunit protein polyclonal antibody prepared by this method could be used for the further researches of the biological function of the XOD in the chicken.
黄嘌呤氧化酶（XOD）是钼羟化酶黄素蛋白家族的成员，在机体嘌呤分解代谢中发挥着重要作用。在本研究中，我们基于从鸡肝脏提取的总RNA，利用RT-PCR和pMD-18-T克隆载体克隆了XOD 37kDa亚基蛋白。将克隆的XOD亚基蛋白基因连接到pET-32a中构建重组质粒pET-XOD。将pET-XOD表达载体转化到宿主细胞Rosetta (DE3) 后，用异丙基-β-D-硫代半乳糖苷（IPTG）成功诱导出重组XOD亚基蛋白（54.8kDa）。以纯化的重组XOD亚基蛋白为抗原制备兔抗血清。用ELISA测定抗血清效价大于1:102,400。Western blot结果表明该抗血清能特异性识别鸡肝脏XOD。免疫组织化学和免疫荧光显示XOD主要存在于鸡肝细胞和近端肾小管上皮细胞的细胞质中。我们的结果表明，通过该方法制备的XOD亚基蛋白多克隆抗体可用于进一步研究鸡体内XOD的生物学功能。
