[1]尚书凤,赵小龙,杨书洋,等.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[J].江苏农业学报,2022,38(05):1286-1297.[doi:doi:10.3969/j.issn.1000-4440.2022.05.016]
　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]
点击复制

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()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
38
期数:
2022年05期
页码:
1286-1297
栏目:
畜牧兽医·水产养殖
出版日期:
2022-10-31

文章信息/Info

Title:
Eukaryotic expression and bioinformatics characteristics of 1α hydroxylase and its differential expression in adult broiler tissues
作者:
尚书凤1; 赵小龙2; 杨书洋1; 郭素芬1; 樊阔1; 李天1; 王琦1
（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）
Author(s):
SHANG Shu-feng1; ZHAO Xiao-long2; YANG Shu-yang1; GUO Su-fen1; FAN Kuo1; LI Tian1; WANG Qi1
(1.College of Bioscience and Engineering, Shaanxi University of Technology, Hanzhong 723000， China;2.Hanzhong Central Hospital, Hanzhong 723000, China)
关键词:
鸡; 细胞色素P450 27C1; 真核表达; 生物信息学
Keywords:
chicken; cytochrome P450 27C1; eukaryotic expression; bioinformatics
分类号:
S831.9
DOI:
doi:10.3969/j.issn.1000-4440.2022.05.016
文献标志码:
A
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。
Abstract:
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.

参考文献/References:

[1]SHUMYANTSEVA V V, KUZIKOV A V, MASAMREKH R A, et al. From electrochemistry to enzyme kinetics of cytochrome P450[J]. Biosensors and Bioelectronics, 2018,121:192-204.
[2]GUENGERICH F P, WATERMAN M R, EGLI M. Recent structural insights into cytochrome P450 function[J]. Trends in Pharmacological Sciences, 2016, 37(8): 625-640.
[3]HUTTON K C, VAUGHN M A, LITTA G, et al. Effect of vitamin D status improvement with 25-hydroxycholecalciferol on skeletal muscle growth characteristics and satellite cell activity in broiler chickens[J]. Journal of Animal Science, 2014, 92(8): 3291-3299.
[4]SHOJADOOST B, BEHBOUDI S, VILLANUEVA A I, et al. Vitamin D3 modulates the function of chicken macrophages[J]. Research in Veterinary Science, 2015, 100: 45-51.
[5]KOTTFEROV J, KORNEKOV B, SIKLENKA P, et al. The effect of Cd and vitamin D3 on the solidity of eggshell[J]. European Food Research and Technology, 2001, 212(2): 153-155.
[6]SHANMUGASUNDARAM R， SELVARAJ R K. Vitamin D-1α-hydroxylase and vitamin D-24-hydroxylase mRNA studies in chickens[J]. Poultry Science, 2012, 91(8): 1819-1824.
[7]YOSHIDA T, YOSHIDA N, NAKAMURA A, et al. Cloning of porcine 25-hydroxyvitamin D3 1α-hydroxylase and its regulation by cAMP in LLC-PK1 cells[J]. Journal of the American Society of Nephrology, 1999, 10(5): 963-970.
[8]UCHIDA E, KAGAWA N, SAKAKI T, et al. Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES[J]. Biochem Biophys Res Commun, 2004, 323(2): 505-511.
[9]陈思航,尚书凤,王杨科,等. 细胞色素P450 3A7羟化维生素D3的功能研究[J]. 基因组学与应用生物学, 2020, 39(1): 30-36.
[10]KUSANO K, KAGAWA N, SAKAGUCHI M, et al. Importance of a proline-rich sequence in the amino-terminal region for correct folding of mitochondrial and soluble microbial P450s[J]. The Journal of Biochemistry, 2001, 129(2): 271-277.
[11]KAZUTOMI K, MASAO S, NORIO K, et al. Microsomal P450s use specific proline-rich sequences for efficient folding, but not for maintenance of the folded structure[J]. Journal of Biochemistry, 2001, 129(2): 259-269.
[12]ANNALORA A J, GOODIN D B, HONG W X, et al. Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism[J]. Journal of Molecular Biology, 2009, 396(2):441-451.
[13]YAMAMOTO K, MASUNO H, SAWADA N, et al. Homology modeling of human 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) based on the crystal structure of rabbit CYP2C5[J]. Journal of Steroid Biochemistry & Molecular Biology, 2004, 89/90(1/2/3/4/5):167-171.
[14]PROSSER D E, GUO Y D, JIA Z C, et al. Structural motif-based homology modeling of CYP27A1 and site-directed mutational analyses affecting vitamin D hydroxylation[J]. Biophysical Journal, 2006, 90(10): 3389-3409.
[15]STRUSHKEVICH N, USANOV S A, PLOTNIKOV A N, et al. Structural analysis of CYP2R1 in complex with vitamin D3[J]. Journal of Molecular Biology, 2008, 380(1):95-106.
[16]TAM B, SINHA S, WANG S M. Combining Ramachandran plot and molecular dynamics simulation for structural-based variant classification: using TP53 variants as model[J]. Computational and Structural Biotechnology Journal, 2020, 18(2): 4033-4039.
[17]刘阳星月，张迪，姚亚亚，等. 大豆过敏原11S球蛋白G2中A2链结合表位的预测[J]. 食品科学, 2018, 39(18): 152-158.
[18]HASEMANN C A, KURUMBAIL R G, BODDUPALLI S S, et al. Structure and function of cytochromes P450: a comparative analysis of three crystal structures[J]. Structure, 1995, 3(1): 41-62.
[19]HANNEMANN F, BICHET A, EWEN K M, et al. Cytochrome P450 systems-biological variations of electron transport chains[J]. Biochimica Et Biophysica Acta General Subjects, 2007, 1770(3): 330-344.
[20]FINNIGAN J D, YOUNG C, COOK D J, et al. Cytochromes P450 (P450s): a review of the class system with a focus on prokaryotic P450s[J]. Advances in Protein Chemistry and Structural Biology, 2020, 122: 290-322.
[21]柳惠琳. Pyroxd2在线粒体的定位及相关机制的研究[D].杭州：杭州师范大学, 2017.
[22]BAE D H, MARINO M, IAFFALDANO B, et al. Design and testing of vector producing HEK 293T cells bearing a genomic deletion of the SV40 T antigen coding region[J]. Methods & Clinical Development, 2020, 18: 631-638.
[23]REN J X, YANG L Y, LI Q L, et al. Global investigation of cytochrome P450 genes in the chicken genome[J]. Genes, 2019, 10(8): 617-633.
[24]GRAY R W, OMDAHL J L, GHAZARIAN J G, et al. 25-hydroxycholecalciferol-1-hydroxylase: subcellular location and properties[J]. Journal of Biological Chemistry, 1972, 247(23): 7528-7532.
[25]CHENG J B, MOTOLA D L, MANGELSDORF D J, et al. De-orphanization of cytochrome P450 2R1[J]. Journal of Biological Chemistry, 2003, 278(39): 38084-38093.
[26]TANG E K Y, TIEU E W, TUCKEY R C, et al. Expression of human CYP27B1 in Escherichiacoli and characterization in phospholipid vesicles[J]. The FEBS Journal, 2012, 279(19): 3749-3761.

相似文献/References:

[1]金崇富,杨智青,陈长宽,等.盐城地方鸡MSTN 基因外显子1 多态性及其与生产性能的相关性分析[J].江苏农业学报,2016,(06):1372.[doi:doi:10.3969/j.issn.1000-4440.2016.06.027]
　JIN Chong-fu,YANG Zhi-qing,CHEN Chang-kuan,et al.Association of MSTN gene with growth and reproductive traits in Yancheng chicken[J].,2016,(05):1372.[doi:doi:10.3969/j.issn.1000-4440.2016.06.027]
[2]金崇富,陈长宽,杨智青,等.鸡IGF1R、IGFBP-3基因SNP位点之间互作效应分析[J].江苏农业学报,2015,(05):1099.[doi:doi:10.3969/j.issn.1000-4440.2015.05.024]
　JIN Chong-fu,CHEN Chang-kuan,YANG Zhi-qing,et al.Genetic interaction effect between SNPs loci of IGF1R and IGFBP-3 genes in chicken[J].,2015,(05):1099.[doi:doi:10.3969/j.issn.1000-4440.2015.05.024]
[3]刘青涛,李银,刘娜,等.H9N2亚型禽流感病毒对鸡的免疫抑制机制[J].江苏农业学报,2018,(02):368.[doi:doi:10.3969/j.issn.1000-4440.2018.02.021]
　LIU Qing-tao,LI Yin,LIU Na,et al.The immunosuppressive mechanism of H9N2 subtype avian influenza virus infection in chickens[J].,2018,(05):368.[doi:doi:10.3969/j.issn.1000-4440.2018.02.021]
[4]黄正洋,王钱保,黄华云,等.鸡let-7b在不同生长时期及不同组织中的表达及其生物信息学分析与靶基因预测[J].江苏农业学报,2022,38(02):429.[doi:doi:10.3969/j.issn.1000-4440.2022.02.017]
　HUANG Zheng-yang,WANG Qian-bao,HUANG Hua-yun,et al.Expression of let-7b in different growth stages and different tissues of chicken and its bioinformatics analysis and target gene prediction[J].,2022,38(05):429.[doi:doi:10.3969/j.issn.1000-4440.2022.02.017]
[5]魏清宇,吴鹏飞,叶红心,等.边鸡快长型与慢长型品系胚胎骨骼肌发育中差异可变剪切[J].江苏农业学报,2023,(02):444.[doi:doi:10.3969/j.issn.1000-4440.2023.02.017]
　WEI Qing-yu,WU Peng-fei,YE Hong-xin,et al.Differential alternative splicing during embryonic skeletal muscle development in fast-growing and slow-growing strains of Bian chicken[J].,2023,(05):444.[doi:doi:10.3969/j.issn.1000-4440.2023.02.017]
[6]朱淼,李欣燃.鸡HSP70家族基因鉴定及功能分析[J].江苏农业学报,2023,(03):770.[doi:doi:10.3969/j.issn.1000-4440.2023.03.018]
　ZHU Miao,LI Xin-ran.Identification and functional analysis of the chicken HSP70 family genes[J].,2023,(05):770.[doi:doi:10.3969/j.issn.1000-4440.2023.03.018]

备注/Memo

备注/Memo:
收稿日期：2022-03-30基金项目：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]作者简介：尚书凤（1978-），女，陕西安康人，博士，讲师，主要研究方向为动物生理生化。(E-mail)shangshf@snut.edu.cn通讯作者：王琦，（Tel）0916-2641576;(E-mail)412704720@qq.com
更新日期/Last Update: 2022-11-07