Prokaryotic Expression, Polyclonal Antibody Preparation and Purification of PPARδ Protein in Nile Tilapia
-
摘要:
目的 为研究尼罗罗非鱼过氧化物酶体增殖物激活受体δ(PPARδ)的表达情况,通过大肠杆菌表达系统表达PPARδ,纯化重组蛋白并获得多克隆抗体。 方法 对PPARδ进行生物信息学分析后设计特异性引物,扩增PPARδ,将其克隆至原核表达载体pET-B2m中,构建重组表达载体;重组质粒转入大肠杆菌B21并用IPTG进行诱导表达,用SDS-PAGE鉴定表达产物。用镍柱纯化重组蛋白后免疫日本大耳兔制备多克隆抗体,用间接ELISA技术检测抗体效价,Western Blot鉴定抗体的特异性。 结果 成功构建了原核表达载体pET-B2m-PPARδ,实现了重组蛋白的原核表达和纯化,重组蛋白以包涵体蛋白和可溶性蛋白2种形式存在,分子量约为90 kD;制备的多克隆抗体效价为1 ∶ 2 048 000,Western Blot检测结果表明该抗体能特异性识别尼罗罗非鱼PPARδ。 结论 成功实现了尼罗罗非鱼PPARδ重组蛋白的融合表达,在大肠杆菌中高效表达后纯化重组蛋白,免疫日本大耳兔获得了效价高、特异性强的多克隆抗体,为尼罗罗非鱼PPARδ的功能研究奠定了基础。 Abstract:Objective The prokaryotic expression of peroxisome proliferator-activated receptor δ (PPARδ) in Nile tilapia was determined for the preparation of a polyclonal antibody against the receptor protein. Method The bioinformatics analysis on PPARδ was conducted to arrive at a primer designed for amplification. The amplified products were subcloned into expression vector pET-B2m to construct recombinant plasmid for transformation into E. coli B21 with IPTG induction. The expressed protein was identified by SDS-PAGE and purified with a Ni-NTA prior to immunization on Japanese white rabbits. A polyclonal antibody with its titer determined by ELISA and specificity analyzed by the western blotting was obtained. Result The prokaryotic expression vector pET-B2m-PPARδ was successfully constructed. The recombinant protein with molecular weight of 90 KD was expressed in both inclusion body and soluble proteins. The titer of the rabbit anti-PPARδ polyclonal antibody was 1 ∶ 2 048 000. The western blot confirmed that the antibody reacted specifically with PPARδ protein. Conclusion This study successfully expressed and purified the PPARδ recombinant protein of Nile tilapia, and subsequently, obtained the polyclonal antibodies with high specificity and efficacy that could be used for further research on PPARδ in Nile tilapia. -
Key words:
- Nile Tilapia /
- PPARδ /
- prokaryotic expression /
- polyclonal antibody
-
图 5 Western blotting分析PPARδ多克隆抗体的特异性
注:M:蛋白标准;1:25 ng PPARδ重组蛋白(G480);2:10 ng PPARδ重组蛋白(G480);3:25 ng PPARδ重组蛋白(G479);4:10 ng PPARδ重组蛋白(G479);5:阴性对照(G480);6:阴性对照(G479)。其中,G480和G479是日本大耳兔的实验编号。
Figure 5. Specificity of PPARδ polyclonal antibody by western blotting
Note: M: marker; 1: 25 ng PPARδ recombinant protein (G480); 2: 10 ng PPARδ recombinant protein (G480); 3: 25 ng PPARδ recombinant protein (G479); 4: 10 ng PPARδ recombinant protein (G479); 5: negative control (G480); and, 6: negative control (G479). G480 and G479 are codes of immunized Japanese white rabbits.
-
[1] ZHENG J L, ZHUOM Q, LUO Z, et al. Peroxisome proliferator-activated receptor gamma (PPARγ) in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA expression and transcriptional regulation by insulin in vivo and in vitro [J]. General and Comparative Endocrinology, 2015, 212: 51−62. doi: 10.1016/j.ygcen.2014.12.020 [2] BATISTA-PINTO C, RODRIGUES P, ROCHA E, et al. Identification and organ expression of peroxisome proliferator activated receptors in brown trout (Salmo trutta f. fario) [J]. Biochimica et Biophysica Acta, 2005, 1731(2): 88−94. doi: 10.1016/j.bbaexp.2005.09.001 [3] 王靖天, 曹纬楠, 霍忠明, 等. 菲律宾蛤仔PPAR基因家族特征分析及干露胁迫下表达变化 [J]. 大连海洋大学学报, 2019, 34(2):168−178.WANG J T, CAO W N, HUO Z M, et al. Characterization and distinct expression patterns of PPAR genes in Manila clam Ruditapes philippinarum in response to air exposure [J]. Journal of Dalian Ocean University, 2019, 34(2): 168−178.(in Chinese) [4] VAN DIEPEN J A, JANSEN P A, BALLAK D B, et al. PPAR-alpha dependent regulation of vanin-lmediates hepatic lipid metabolism [J]. Journal of hepatology, 2014, 61(2): 366−372. doi: 10.1016/j.jhep.2014.04.013 [5] 高 俊, 徐钢春, 杜富宽, 等. 刀鲚PPARγ基因的cDNA克隆及其应激应答 [J]. 中国水产科学, 2019, 26(2):242−250.GAO J, XU G C, DU F K, et al. Molecular cloning and stress response of PPARγ in Coilia nasus [J]. Journal of Fishery Sciences of China, 2019, 26(2): 242−250.(in Chinese) [6] ROSEN E D, WALKEY C J, PUIGSERVER P, et al. Transcriptional regulation of adipogenesis [J]. Genes & development, 2000, 14(11): 1293−1307. [7] 陈 娜, 吴英良. PPARδ的结构及其生物学功能与疾病 [J]. 中国药理学通报, 2006, 22(9):1035−1038. doi: 10.3321/j.issn:1001-1978.2006.09.003CHEN N, WU Y L. A research on the structure and biological functions of PPARδ and its relationship with diseases [J]. Chinese Pharmacological Bulletin, 2006, 22(9): 1035−1038.(in Chinese) doi: 10.3321/j.issn:1001-1978.2006.09.003 [8] XU H E, LAMBERT M H, MONTANA V G, et al. Molecular recognition of fatty acids by peroxisome proliferator-activated receptors [J]. Molecular cell, 1999, 3(3): 397−403. doi: 10.1016/S1097-2765(00)80467-0 [9] BARISH G D, NARKAR V A, EVANS R M. PPARδ: a dagger in the heart of the metabolic syndrome [J]. Journal of Clinical Investigation, 2006, 116(3): 590−597. doi: 10.1172/JCI27955 [10] 方玲玲, 陈 刚, 王忠良, 等. 卵形鲳鲹PPARα基因cDNA序列的克隆、组织表达及生物信息学分析 [J]. 广东海洋大学学报, 2015, 35(4):1−9. doi: 10.3969/j.issn.1673-9159.2015.04.001FANG L L, CHEN G, WANG Z L, et al. Molecular cloning and expression distribution and bioinformatics analysis of Peroxisome Proliferator Activated Receptors-α in Trachinotus ovatus [J]. Journal of Guangdong Ocean University, 2015, 35(4): 1−9.(in Chinese) doi: 10.3969/j.issn.1673-9159.2015.04.001 [11] 钱 伦, 钱云霞, 童丽娟. 大黄鱼PPAR β全长cDNA的克隆和组织表达 [J]. 生物学杂志, 2010, 27(6):1−4. doi: 10.3969/j.issn.1008-9632.2010.06.001QIAN L, QIAN Y X, TONG L J. Cloning of full-length cDNA and RT-PCR expression analysis of PPARβ in Larimichthys crocea [J]. Journal of Biology, 2010, 27(6): 1−4.(in Chinese) doi: 10.3969/j.issn.1008-9632.2010.06.001 [12] 贾成霞, 张照斌, 张清靖, 等. 虹鳟PPARα基因克隆、序列分析及其组织表达分布 [J]. 中国水产科学, 2012, 19(4):707−714.JIA C X, ZHANG Z B, ZHANG Q J, et al. Cloning, sequence analysis, and expression distribution of PPARα from rainbow trout [J]. Journal of Fishery Sciences of China, 2012, 19(4): 707−714.(in Chinese) [13] 钱云霞, 杨孙孝, 梁 洪, 等. 鲈PPARγ基因的克隆、组织表达及其抗体制备 [J]. 水产学报, 2010, 34(8):1156−1164.QIAN Y X, YANG S X, LIANG H, et al. Gene cloning, tissue expression and preparation of monoclonal antibody of Lateolabrax japonicus PPARγ [J]. Journal of Fisheries of China, 2010, 34(8): 1156−1164.(in Chinese) [14] ZHAO Y, GUL Y, LI S, et al. Cloning, identification and accurate normalization expression analysis of PPARα, gene by GeNorm in Megalobrama amblycephala [J]. Fish & Shellfish Immunology, 2011, 31: 462−468. [15] IBABE A, GRABENBAUER M, BAUMGART E, et al. Expression of peroxisome proliferator-activated receptors in zebrafish (Danio rerio) [J]. Histochemie, 2002, 118: 231−239. [16] 潘传燕, 林 勇, 冯鹏霏, 等. 尼罗罗非鱼Hsc70的原核表达和多克隆抗体制备 [J]. 浙江农业学报, 2019, 31(8):1272−1279. doi: 10.3969/j.issn.1004-1524.2019.08.07PAN C Y, LIN Y, FENG P F, et al. Prokaryotic expression and polyclonal antibody preparation of Hsc70 in Nile Tilapia [J]. Acta Agriculturae Zhejiangensis, 2019, 31(8): 1272−1279.(in Chinese) doi: 10.3969/j.issn.1004-1524.2019.08.07 [17] 张永德, 林 勇, 冯鹏霏, 等. 尼罗罗非鱼Lck多克隆抗体的制备及鉴定 [J]. 南方农业学报, 2018, 49(11):2304−2310. doi: 10.3969/j.issn.2095-1191.2018.11.27ZHANG Y D, LIN Y, FENG P F, et al. Preparation and identification of Lck polyclonal antibody in Nile tilapia [J]. Journal of Southern Agriculture, 2018, 49(11): 2304−2310.(in Chinese) doi: 10.3969/j.issn.2095-1191.2018.11.27 [18] CHO H K, KONG H J, NAM B H, et al. Molecular cloning and characterization of olive flounder(Paralichthys olivaceus)peroxisome proliferator-activated receptor gamma [J]. Gen Comp Endocrinol, 2009, 163(3): 251−258. doi: 10.1016/j.ygcen.2009.04.018 [19] 陈 亮, 梁旭方, 瞿春梅, 等. 草鱼过氧化物酶体增殖物激活受体(PPAR)基因cDNA序列的克隆及其组成型表达 [J]. 暨南大学学报: 自然科学版, 2011, 32(1):80−87.CHEN L, LIANG X F, ZHAI C M, et al. Molecular cloning of peroxisome proliferator activated receptors and constitutive expression in grass carp (Ctenopharyngodon idellus) [J]. Journal of Jinan University(Natural Science), 2011, 32(1): 80−87.(in Chinese) [20] 王净修, 高章照, 姜永厚, 等. 猪圆环病毒2型ORF4基因的原核表达及多克隆抗体的制备 [J]. 浙江农业学报, 2014, 26(1):34−39. doi: 10.3969/j.issn.1004-1524.2014.01.07WANG J X, GAO Z Z, JIANG Y H, et al. Prokaryotic expression of the PCV2 ORF4 gene and preparation of polyclonal antibodies against ORF4 [J]. Acta Agriculturae Zhejiangensis, 2014, 26(1): 34−39.(in Chinese) doi: 10.3969/j.issn.1004-1524.2014.01.07 [21] 何玲玲. 人巨细胞病毒UL146基因编码蛋白vCXCL-1的重组表达及其多克隆抗体的制备和鉴定[D]. 广州: 暨南大学, 2018.HE L L. Recombinant expression of human cytomegalovirus UL146 gene encoding protein vCXCL-1 and its polyclonal antibody preparation and identification[D]. Guangzhou: Jinan University, 2018. (in Chinese) [22] 张 旻, 郑晓聪, 林祥梅. 锦鲤疱疹病毒ORF1基因的克隆、表达及多克隆抗体的制备 [J]. 中国农学通报, 2012, 28(5):98−102. doi: 10.3969/j.issn.1000-6850.2012.05.019ZHANG M, ZHENG X C, LIN X M. PCR amplification prokaryotic expression and polyclonal antibody preparation of Koi Herpesvirus Virus ORF1 Gene [J]. Chinese Agricultural Science Bulletin, 2012, 28(5): 98−102.(in Chinese) doi: 10.3969/j.issn.1000-6850.2012.05.019 [23] 李素一, 张丽娟, 陈 华, 等. 创伤弧菌铁调基因fur的原核表达及多克隆抗体制备 [J]. 福建农业学报, 2016, 31(9):912−916.LI S Y, ZHANG L J, CHEN H, et al. Prokaryotic expression and polyclonal antibody for Ferric Uptake Regulator Prepared from Vibrio vulnificus FJ03-x2 [J]. Fujian Journal of Agricultural Sciences, 2016, 31(9): 912−916.(in Chinese)