1α羟化酶的真核表达和生物信息学特征及其在成年肉鸡组织中的差异表达  

Eukaryotic expression and bioinformatics characteristics of 1αhydroxylase and its differential expression in adult broiler tissues

作　　者：尚书凤[1] 赵小龙[2] 杨书洋 郭素芬[1] 樊阔 李天 王琦[1] SHANG Shu-feng;ZHAO Xiao-long;YANG Shu-yang;GUO Su-fen;FAN Kuo;LI Tian;WANG Qi(College of Bioscience and Engineering,Shaanxi University of Technology,Hanzhong 723000,China;Hanzhong Central Hospital,Hanzhong 723000,China)

机构地区：[1]陕西理工大学生物科学与工程学院,陕西汉中723000 [2]汉中市中心医院,陕西汉中723000

出　　处：《江苏农业学报》2022年第5期1286-1297,共12页Jiangsu Journal of Agricultural Sciences

基　　金：陕西省教育厅专项科研计划项目(16JK1148);陕西理工大学人才启动项目[SLGQ13(2)-24]。

摘　　要：旨在构建1羟化酶(CYP27C1)真核表达载体,在293T细胞中重组表达CYP27C1-mycHis,探究cyp27c1基因在成年鸡不同组织中的表达水平,明确1α-羟化酶三级结构特征、与底物识别和活性中心底物结合相关的关键氨基酸。将合成的cyp27c1的开放阅读框(ORF)插入pMD18-T质粒,并将该质粒命名为pMD-cyp27c1。以改造后的质粒为模板,通过PCR扩增cyp27c1 ORF,将其插入pcDNA^(TM)3.1,构建真核表达载体pcDNA-cyp27c1。然后分别用pcDNA-cyp27c1及空载体pcDNA瞬时转染293T细胞,48 h后收集细胞,提取线粒体,用Bradford法测定蛋白质的质量浓度。1μg线粒体用于12%聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,以myc抗体作为一抗检测重组CYP27C1在293T细胞中的表达水平。利用实时荧光定量PCR检测cyp27c1在成年鸡组织中的表达水平。通过生物信息学分析CYP27C1的理化特性,并预测其亚细胞定位,利用同源建模、多序列同源比对和分子对接法研究CYP27C1的空间结构特征以及与底物(25-OH D_(3))结合和识别相关的关键氨基酸。测序结果显示,1608 bp编码区插入到pcDNA^(TM)3.1,并成功在其3′端融合了载体表达标签myc-His。在重组表达CYP27C1-mycHis的293T细胞线粒体中检测到相对分子质量为58000的特异性条带。实时荧光定量PCR结果表明,cyp27c1基因在鸡肝、肾、胸肌、腿肌、小肠、胸腺、脾、肾上腺、睾丸、卵巢等组织中均有表达,并且相对表达量存在组织差异性。cyp27c1基因编码1个由536个氨基酸组成的细胞色素P450,亚细胞定位于线粒体上,成熟蛋白质N-末端起始于V62。同源建模结果显示,CYP27C1中α螺旋、β折叠的氨基酸数量分别占蛋白质中总氨基酸数量的49.15%、5.90%,具有与其他线粒体细胞色素P450相似的开放式三级结构。多序列比对和分子对接结果表明,C482、R135与血红素形成较强的氢键;A136、K83是与25-OH D_(3)识别的关键氨基酸;活性中心L343、D347是与25-OH DThe aim of this study was to construct pMD-cyp27c1 eukaryotic expression vector,express recombinant CYP27C1-mycHis in 293T cells,explore the expression levels of cyp27c1 in different tissues of adult chickens,and confirm the tertiary structure and the key amino acids involved in identifying and binding substrate in active-center.The open reading frame(ORF)of cyp27c1 produced artificially was inserted into pMD18-T,which was named pMD-cyp27c1.The ORF of cyp27c1 was amplified by PCR using the modified plasmids as templates,and subsequently inserted into pcDNA^(TM)3.1 to construct pcDNA-cyp27c1 eukaryotic expression vector.The 293T cells were transiently transfected with pcDNA-cyp27c1 and empty vector pcDNA,respectively.After 48 hours,the cells were collected.The mitochondria were extracted,and the protein concentration was determined by Bradford method.The 1μg mitochondria were used for 12%sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)separation.Herein,myc antibody was used to estimate the expression of recombinant CYP27C1 in 293T cells.The expression level of cyp27c1 in adult chicken was detected by real-time fluorescence quantitative PCR.The physicochemical properties of CYP27C1 were analyzed by bioinformatics,and its subcellular localization was predicted.The structure characteristics and key amino acids involved in identifying and binding substrate(25-OH D_(3))were confirmed by homology modeling,molecular docking and multi-sequence homology alignment.The results showed that encoding sequence of 1608 bp was inserted into pcDNA^(TM)3.1.The expression tag myc-His was successfully fused at the 3′end.A specific band with a relative molecular weight of 58000 was detected in the mitochondria of 293T cells expressing recombinant CYP27C1-mycHis.The results of real-time fluorescence quantitative PCR showed that cyp27c1 gene was expressed in liver,kidney,chest muscle,thigh,small intestine,thymus,spleen,adrenal gland,testicles and ovary.And mRNA expression levels displayed tissue variability.The cyp

关 键 词：鸡 细胞色素P45027C1 真核表达 生物信息学 

分 类 号：S831.9[农业科学—畜牧学]