SHANG Shu-feng,ZHAO Xiao-long,YANG Shu-yang,et al.Eukaryotic expression and bioinformatics characteristics of 1α hydroxylase and its differential expression in adult broiler tissues[J].,2022,38(05):1286-1297.[doi:doi:10.3969/j.issn.1000-4440.2022.05.016]





Eukaryotic expression and bioinformatics characteristics of 1α hydroxylase and its differential expression in adult broiler tissues
SHANG Shu-feng1ZHAO Xiao-long2YANG Shu-yang1GUO Su-fen1FAN Kuo1LI Tian1WANG Qi1
(1.College of Bioscience and Engineering, Shaanxi University of Technology, Hanzhong 723000, China;2.Hanzhong Central Hospital, Hanzhong 723000, China)
细胞色素P450 27C1真核表达生物信息学
chickencytochrome P450 27C1eukaryotic expressionbioinformatics
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pcDNATM3.1,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。1 μg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 D3)结合和识别相关的关键氨基酸。测序结果显示,1 608 bp编码区插入到pcDNATM3.1,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58 000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 D3识别的关键氨基酸;活性中心L343、D347是与25-OH D3结合的关键氨基酸。本研究成功构建了CYP27C1真核表达载体,并在293T细胞中超表达CYP27C1,确定了CYP27C1的三级结构和与底物识别及结合相关的关键氨基酸,为后续深入研究CYP27C1结构、功能和催化机制提供了基础。
The 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 pcDNATM3.1 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 1 μg 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 D3) were confirmed by homology modeling, molecular docking and multi-sequence homology alignment. The results showed that encoding sequence of 1 608 bp was inserted into pcDNATM3.1. The expression tag myc-His was successfully fused at the 3′end. A specific band with a relative molecular weight of 58 000 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 cyp27c1 encoded a mitochondrial cytochrome P450 composed by 536 amino acids. The N-terminal of mature protein started at V62. The alpha-helixes and beta-sheets accounted for 49.15% and 5.90% of mature protein, respectively. CYP27C1 protein had a typical open tertiary structure similar to other mitochondrial cytochrome P450. The results of molecular docking and multi-sequence homology alignment indicated that C482 and R135 formed strong hydrogen bonds with heme. A136 and K83 were key amino acids recognizing 25-OH D3. L343 and D347 in the active centers were key amino acids interacting 25-OH D3. In this study, the CYP27C1 eukaryotic expression vector is successfully constructed, and CYP27C1 is expressed in 293T cells. The tertiary structure and the key amino acids involved in binding and recognizing the substrate are determined. These results provide a basis for further study on the structure, function and catalytic mechanism of CYP27C1.


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更新日期/Last Update: 2022-11-07