期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

抗人骨桥蛋白单克隆抗体的制备及鉴定 被引量：5

原文传递
导出
摘要 目的:制备抗人骨桥蛋白(hOPN)的单克隆抗体(mAb),用于其功能研究。方法:构建OPN的原核表达载体pTriEx-1-hONP载体,转化Tuner(DE3)placI感受态大肠杆菌,用IPTG进行诱导表达。利用镍柱亲和层析纯化OPN蛋白,纯化蛋白经超滤浓缩洗涤后即为免疫原。以重组纯化的OPN蛋白为免疫原,免疫BALB/c小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞Sp2/0进行常规融合,通过有限稀释法进行克隆和间接ELISA筛选,获得分泌鼠抗人OPN蛋白mAb的杂交瘤细胞株,通过ELISA、Western blot等方法检测其特异性,并用免疫组化方法检测了正常月经周期子宫内膜OPN的表达。结果:pTriEx-1-hONP表达的OPN蛋白主要为可溶性形式,经镍柱纯化后,蛋白纯度可达85%以上。纯化的OPN重组蛋白免疫小鼠后经融合筛选,得到2株稳定分泌抗人OPN的mAb杂交瘤细胞株,分别命名为1E7和5B7,两株mAb的免疫球蛋白亚类分别IgG1和IgG2a。通过ELISA和Western blot等方法鉴定,抗OPN的mAb能与OPN蛋白特异性结合。通过免疫组织化学方法对不同时期的正常子宫内膜的OPN的检测表明,子宫内膜腺上皮在增生期、分泌早期,OPN呈弱阳性表达;分泌中、晚期OPN呈强阳性表达。结论:以纯化的重组hOPN为免疫原成功制备了鼠抗hOPN的mAb,并初步进行了应用,为研究hOPN的功能打下了良好基础。
作者 周群芳 鞠吉雨 郎景和
机构地区 民航总医院妇产科 潍坊医学院免疫学教研室山东省高等学校免疫学重点实验室
出处 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2009年第1期62-64,共3页 Chinese Journal of Cellular and Molecular Immunology
基金 国家自然科学基金资助项目(39830350)
关键词 骨桥蛋白 原核表达 单克隆抗体
分类号 R392.11 [医药卫生—免疫学]
  • 相关文献

参考文献7

  • 1Khan SA, Cook AC, Kappil M, et al. Enhanced cell surface CD44 variant( v6, v9 )expression by osteopontin in breast cancer epithelial ceils facilitates tumor cell migration: Novel post transcriptional, post translational regulation [ J ]. Clin Exp Metastasis, 2005, 22 ( 8 ) : 663 - 673.
  • 2Rittling SR, Chambers AF. Role of osteopontin in tumor progression [J]. Br J Cancer, 2004, 90(10) : 1877 - 1551.
  • 3Kim JH, Skates SJ, Uede T, et al. Osteopontin as a potential diagnostic biomarker for ovarian cancer[ J]. JAMA, 2002, 287 ( 13 ) : 1671 - 1679.
  • 4White FJ, Ross JW, Joyce MM, et al. Steroid regulation of cell specitic secreted phosphoprotein 1 (osteopontin)expression in the pregnant porcine uterus[J].Biol Reprod, 2005, 73 (6) : 1294 - 1301.
  • 5Weintraub AS, Lin X, Itskovich VV, et al. Prenatal detection of embryo resorption in osteopontin deficient mice using serial noninvasive magnetic respane microscopy[ J]. Pediatr Res, 2004, 55 (3) : 419 - 424.
  • 6Helluint O, Chan C, Vilaire G, et al. The activation state of αvβ3 regulates platelet and lymphocyte adhesion to intact and thrombin cleaved osteopontin [ J ]. J Biol Chem, 2000, 275 (24) : 18337 - 18343.
  • 7马彩虹,陈贵安,韩劲松,齐永芬.骨桥蛋白在人正常月经周期子宫内膜的表达[J].生殖医学杂志,2006,15(1):1-5. 被引量：7

二级参考文献12

  • 1Weber GF,Cantor H. The immunology of Eta-1/Osteopontin[J]. Cytokine Growth Factor Rev,1996, 7(3):241-248.
  • 2Oates AJ, Barraelough R, Rudland PS. The role of osteopontin in tumorigenesis and metastasis [J]. Invasion Metastasis, 1997,17(1):1-15.
  • 3Young MF, Kerr JM, Termine JD, et al. cDNAcloning,mRNA distribution and hetergeneity, chromosomal location,and RFLP analysis of human osteopontin (OPN) [J].Genomics, 1990,7 ( 10 ) : 491-502.
  • 4Apparao KB, Murray MJ, Fritz MA, et al. Osteopontin and its receptor alphavbeta(3) integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially[J]. J Clin Endocrinol Metab, 2001, 86 (10) :4991-5000.
  • 5Johnson GA, Burghardt RC, Spencer TE, et al. Ovine osteopontin: Ⅱ. Osteopontin and alpha (v) beta (3) integrin expression in the uterus and conceptus during the periimplantation period[J]. Biol Reprod, 1999,61 (4) : 892-899.
  • 6Qi YF, Bu DF, Niu daD, et al. Effeets of different peptide fragments derived from proadrenomedullin on gene expression of adrenomedullin gene[J]. Peptides,2002,23(3):1141-1147.
  • 7von-Wolff M, Strowitzki T, Beeker V, et al. Endometrial osteopontin, a ligand of beta3-integrin, is maximally expressed around the time of the "implantation window"[J].Fertil Steril, 2001, 76(4) :775-781.
  • 8Rout UK, Wang J, Paria BC, et al. Alpha5betal, alph Vbeta3 and the platelet-associated integrin alphaⅡbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophohlast cells[J]. Dev Biol, 2004, 268(1):135-151.
  • 9Senger DR, Ledbetter SR, Claffey KP, et al. Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving ανβ3 integrin, osteopontin and thrombin[J].Am J Pathol,1996,149(1) :293-305.
  • 10Somkuti SG, Yuan LW, Fritz MA, et al. Epidermal growth factor and sex steroids dynamically regulate a marker of endometrial receptivity in Ishikawa cells[J]. J Clin Endocrinol Metab, 1997,82(7) :2192-2197.

共引文献6

同被引文献44

  • 1马彩虹,陈贵安,韩劲松,齐永芬.骨桥蛋白在人正常月经周期子宫内膜的表达[J].生殖医学杂志,2006,15(1):1-5. 被引量：7
  • 2朱耀魁,夏明翰,王晓玉,潘观玉,罗新.骨桥蛋白在子宫内膜异位症患者异位和在位内膜中的表达[J].中华妇产科杂志,2006,41(5):343-344. 被引量：18
  • 3Lopes JE, Torgerson TR, Schubert LA, et al. Analysis of FOXP3 reveals multiple domains required for its function as a transcriptional repressor[J]. J Immunol, 2006, 177(5) : 3133 -3142.
  • 4Carlsson P, Mablapuu M. Forkhead transcription factors: key players in development and metabolism[J].Dev Biol, 2002, 250( 1 ) : 1 - 23.
  • 5Sakaguchi S, Wing K, Miyara M. Regulatory T cells-a brief history and perspective[ J]. Eur J Immunol, 2007, 37 (Suppl 1 ) : S116 - 123.
  • 6Bennett CL, Christie J, Ramsdell F, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3[J]. Nat Genet, 2001,27(1) : 20 - 21.
  • 7Ochs HD, Gambineri E, Torgerson TR. IPEX, FOXP3 and regulatory T-cells: a model for autoimmunity [ J]. Immunol Res, 2007, 38 (1 - 3): 112-121.
  • 8Lin W, Haribhai D, Relland LM, et al. Regulatory T cell development in the absence of functional Foxp3 [ J ]. Nat Immunol, 2007, 8 (4) : 359 -368.
  • 9Wan YY, Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression [ J ]. Nature, 2007, 445(7129) : 766 -770.
  • 10Shariff MI, Cox IJ, Gomaa AI, et al. Hepatocellular carcinoma: cur- rent trends in worldwide epidemiology, risk factors, diagnosis and therapeutics[J]. Expt Rev Gastroenterel Hepatol, 2009, 3:353 - 367.

引证文献5

二级引证文献9

细胞与分子免疫学杂志
2009年 第1期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部