Prokaryotic expression of the chicken xanthine oxidase (XOD) subunit and its localization in liver and kidney

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• 作者：Huayuan Lin¹ ; Yanjun Chen¹ ; Qiqi Huang¹ ; Xiaoquan Guo ; ^{Xqguo20720@aliyun.com" class="auth_mail" title="E-mail the corresponding author} ; Ping Liu ; ^{pingliujx@163.com" class="auth_mail" title="E-mail the corresponding author} ; Weilian Liu ; Caiying Zhang ; Huabin Cao ; Guoliang Hu
• 关键词：Xanthine oxidase ; Chicken ; ELISA ; Western blot ; Immunohistochemistry ; Immunofluorescence
• 刊名：International Journal of Biological Macromolecules
• 出版年：2016
• 出版时间：June 2016
• 年：2016
• 卷：87
• 期：Complete
• 页码：341-347
• 全文大小：3068 K

文摘

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 37 kDa 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 (54.8 kDa) 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.