|本期目录/Table of Contents|

 REN Dongsheng,GUAN Jiafa,LI Dehua,et al.Cloning, Expression, Purification and Polyclonal Antibody Preparation of Human Apolipoprotein A5[J].Chinese Journal of Applied & Environmental Biology,2009,15(01):95-99.[doi:10.3724/SP.J.1145.2009.00095]





Cloning, Expression, Purification and Polyclonal Antibody Preparation of Human Apolipoprotein A5
1中国科学院成都生物研究所 成都 610041
2中国科学院研究生院 北京 100049
3成都地奥制药集团有限公司 成都 610041
REN Dongsheng GUAN Jiafa LI Dehua LIANG Bo WU Qiaqing
1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
2Graduate University of Chinese Academy of Sciences, Beijing 100049, China
3Chengdu Di’ao Pharmaceutical Group Co., Ltd., Chengdu 610041, China
apolipoprotein A5 prokaryotic expression fermentation purification antibody
Q78 : Q592
人载脂蛋白A5 (ApoA5)是血浆三酰甘油水平的重要决定因素之一,与心血管疾病密切相关,因此获得高纯度
的载脂蛋白A5及其抗体具有重要的意义. 本文利用PCR技术,从人肝癌细胞系7721的cDNA中扩增出ApoA5基因,并克
隆到pThioHisD载体中导入大肠杆菌Escherichia coli BL21 (DE3)中进行表达. 经优化条件,发酵得到高效表达的融合蛋
白. 利用融合蛋白上的一段组氨酸序列,用镍离子亲和柱进行纯化,得到纯度大于95%的融合蛋白. 使用纯化蛋白免
疫新西兰大耳兔,得到多克隆抗体,Western印迹结果显示此多克隆抗体能与ApoA5特异性结合. 图6 参28
Apolipoprotein A5 (ApoA5) gene has been shown to play an important role in determining plasma triglyceride
concentrations in humans, and is a major independent risk factor of cardiovascular disease. It is important to obtain the purified ApoA5 and antiserum. In this paper, full-length ApoA5 gene was amplified from human hepatoma cell line 7721 cDNA, and cloned into expression plasmid pThioHisD named as pTh-ApoA5. The pTh-ApoA5 was introduced into Escherichia coli BL21 (DE3). After optimizing its fermentation condition, recombinant ApoA5 was highly expressed. The recombinant ApoA5 protein that contained a 6-His sequence was separated by Ni2+ affinity column and the purity of recombinant ApoA5 was more than 95%. The purified recombinant ApoA5 protein was employed for immunizing rabbits and the antiserum was obtained. Western blot showed that the antibody was specific. Fig 6, Ref 28


 1 Pennacchio LA, Olivier M, Hubacek JA, Cohen JC, Cox DR, Fruchart JC, Krauss RM, Rubin EM. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing. Science, 2001, 294 (5540): 169~173
2 van der Vliet HN, Sammels MG, Leegwater AC, Levels JH, Reitsma PH, Boers W, Chamuleau RA. Apolipoprotein A-V: A novel apolipoprotein associated with an early phase of liver regeneration. J Biol Chem, 2001, 276 (48): 44512~44520
3 Brien PJ, Alborn WE, Sloan JH, Ulmer M, Boodhoo A, Knierman MD, Schultze AE, Konrad RJ. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins. Clin Chem, 2005, 51 (2): 351~359
4 van der Vliet HN, Schaap FG, Levels JH, Ottenhoff R, Looije N, Wesseling JG, Groen AK, Chamuleau RA. Adenoviral overexpression of apolipoprotein A-V reduces serum levels of triglycerides and cholesterol in mice. Biochem Biophys Res Commun, 2002, 295 (5): 1156~1159
5 van Dijk KW, Rensen PC, Voshol PJ, Havekes LM. The role and mode of action of apolipoproteins CIII and AV: Synergistic actors in triglyceride metabolism? Curr Opin Lipidol, 2004, 15 (3): 239~246
6 Aouizerat BE, Kulkarni M, Heilbron D, Drown D, Raskin S, Pullinger CR, Malloy MJ, Kane JP. Genetic analysis of a polymorphism in the human apoA-V gene: Effect on plasma lipids. J Lipid Res, 2003, 44 (6): 1167~1173
7 Frunchart-Najib J, Bauge E, Niculescu LS, Pham T, Thomas B, Rommens C, Majd Z, Brewer B, Pennacchio LA, Fruchart JC. Mechanism of triglyceride lowering in mice expressing human apolipoprotein A5. Biochem Biophys Res Commun, 2004, 319 (2): 397~404
8 Schaap FG, Rensen PC, Voshol PJ, Vrins C, van der Vliet HN, Chamuleau RA, Havekes LM, Groen AK, van Dijk KW. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis. J Biol Chem, 2004, 279 (27): 27941~27947
9 Merkel M, Loeffler B, Kluger M, Fabig N, Geppert G, Pennacchio LA, Laatsch A, Heeren J. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase. J Biol Chem, 2005, 280 (22): 21553~21560
10 Marçais C, Verges B, Charrière S, Pruneta V, Merlin M, Billon S, Perrot L, Drai J, Sassolas A, Pennacchio LA, Frunchart-Najib J, Fruchart JC, Durlach V, Moulin P. Apoa5 Q139X truncation predisposes to late-onset hyperchylomicronemia due to lipoprotein lipase impairment. J Clin Invest, 2005, 115 (10): 2862~2869
11 Weinberg RB, Cook VR, Beckstead JA, Martin DD, Gallagher JW, Shelness GS, Ryan RO. Structure and interfacial properties of human apolipoprotein A-V. J Biol Chem, 2003, 278 (36): 34438~34444
12 Liu Q (刘琼), Zhao SP (赵水平). Preparation and clinical application of monoclonal antibodies against human ApoA5. J Clin Res (医学临床研究), 2006, 23 (2): 153~156
13 Talmud PJ. Rare APOA5 mutations-clinical consequences, metabolic and functional effects: An ENID review. Atherosclerosis, 2007, 194 (2): 287~292
14 Bai Y (白彧), Gou XP (苟小平), Xu Y (徐莺), Yan F (颜钫), Tang L (唐琳), Chen F (陈放). Cloning of a differentially expressed gene in rice sperm cells and preparation of antiserum against expressed product. Chin J Appl Environ Biol (应用与环境生物学报), 2002, 8 (4): 378~382
15 Hokanson JE, Austin MA. Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: A meta-analysis of population-based prospective studies. J Cardiovasc Risk, 1996, 3 (2): 213~219
16 Cullen P. Evidence that triglycerides are an independent coronary heart disease risk factor. Am J Cardiol, 2000, 86 (9): 943~949
17 Fruchart JC, Duriez P. HDL and triglyceride as therapeutic targets. Curr Opin LipidOl, 2002, 13 (6): 605~616
18 Groenendijk M, Cantor RM, de Bruin TW, Dallinga-Thie GM. The apoAI-CIII-AIV gene cluster. Atherosclerosis, 2001, 157 (1): 1~11
19 Shoulders CC, Harry PJ, Lagrost L, White SE, Shah NF, North JD, Gilligan M, Gambert P, Ball MJ. Variation at the apoAI/CIII/AIV gene complex is associated with elevated plasma levels of apoCIII. Atherosclerosis, 1991, 87 (2~3): 239~247
20 Bainton D, Miller NE, Bolton CH, Yarnell JW, Sweetnam PM, Baker LA, Lewis B, Elwood PC. Plasma triglyceride and high density lipoprotein cholesterol as predictors of ischemic heart disease in British men: The Caerphily and Speedwell Collaborative Heart Disease Studies. Br Heart J, 1992, 68 (1): 60~66
21 Schachter NS, Hayek T, Leff T, Smith JD, Rosenberg DW, Walsh A, Ramakrishnan R, Goldberg IJ, Ginsberg HN, Breslow JL. Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice. J Clin Invest, 1994, 93 (4): 1683~1690
22 Baroukh N, Bauge E, Akiyama J, Chang J, Afzal V, Fruchart JC, Rubin EM, Fruchart-Najib J, Pennacchio LA. Analysis of apolipoprotein A5, C3, and plasma triglyceride concentrations in genetically engineered mice. Arterioscler Thromb Vasc Biol, 2004, 24 (7): 1297~1302
23 Garelnabi M, Selvarajan K, Litvinov D, Santanam N, Parthasarathy S. Dietary oxidized linoleic acid lowers triglycerides via APOA5/APOCIII dependent mechanisms. Atherosclerosis, 2008, 199 (2): 304~309
24 Klos KL, Hamon S, Clark AG, Boerwinkle E, Liu K, Sing CF. APOA5 polymorphisms influence plasma triglycerides in young, healthy African Americans and whites of the CARDIA study. J Lipid Res, 2005, 46 (3): 564~571
25 Elosua R, Ordovas JM, Cupples L A, Lai CQ, Demissie S, Fox CS, Polak J F, Wolf PA, D’Agostino RB, O’Donnell CJ. Variants at the APOA5 locus, association with carotid atherosclerosis, and modification by obesity: The Framingham Study. J Lipid Res, 2006, 47 (5): 990~996
26 Corella D, Lai CQ, Demissie S, Cupples LA, Manning AK, Tucker KL, Ordovas JM. APOA5 gene variation modulates the effects of dietary fat intake on body mass index and obesity risk in the Framingham Heart Study. J Mol Med, 2007, 85 (2): 119~128
27 Ishihara M, Kujiraoka T, Iwasaki T, Nagano M, Takano M, Ishii J, Tsuji M, Ide H, Miller IP, Miller NE, Hattori H. A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration. J Lipid Res, 2005, 46 (9): 2015~2022
28 Liu Q (刘琼), Zhao SP (赵水平). Expression and preparation of polyclonal antibodies against human apolipoprotein in A5 and tissue distribution. Chin J Pract Intern Med (中国实用内科学), 2006, 26 (16): 1275~1278


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更新日期/Last Update: 2009-03-05