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人UL16结合蛋白3的原核表达及其多克隆抗体制备 被引量：4

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摘要 目的:表达和纯化人UL16结合蛋白3(ULBP3)胞外段融合蛋白,制备兔抗人ULBP3多克隆抗体。方法:利用PCR技术获得人ULBP3分子胞外段的基因编码序列,并将其亚克隆至原核表达载体pQE30,构建出重组表达质粒pQE30-ULBP3(aa30-171)。测序鉴定正确后转化入大肠杆菌感受态细胞M15,经IPTG 30℃诱导4 h后,SDS-PAGE显示融合蛋白(表达产物)以包涵体形式存在。用镍柱对融合蛋白进行纯化,将纯化的蛋白免疫新西兰白兔,采用流式细胞术(FCM)、Western blot法和免疫组化染色对所得多克隆抗体的生物学特性进行鉴定。结果:成功表达及纯化了人ULBP3(aa30-171)重组蛋白。结果显示该多克隆抗体能够识别大肠癌细胞株COLO205表面表达的天然构象ULBP3分子,而且能与结直肠癌组织细胞的ULBP3分子结合。结论:成功构建了原核表达载体pQE30-ULBP3(aa30-171),并获得高纯度的人pQE30-ULBP3(aa30-171)重组蛋白;成功制备了兔抗人ULBP3分子的多克隆抗体。
作者 秦伟 毛朝明 王胜军 吴蔚 牟笑 许化溪 包丹霞
机构地区 江苏大学附属医院核医学科 江苏大学医学技术学院免疫学与免疫检验学系
出处 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2012年第5期520-523,共4页 Chinese Journal of Cellular and Molecular Immunology
基金 镇江社会发展支撑计划项目(SH2011019 SH2008034) 江苏大学临床医学发展项目(JLY2010157)
关键词 ULBP3 NKG2D 多克隆抗体
分类号 R593.2 [医药卫生—内科学]
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参考文献12

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  • 2Song H, Kim J, Cosman D, et al. Soluble ULBP suppresses natural killer cell activity via down-regulating NKG2D expression[ J]. Cell Immunol, 2006, 239 ( 1 ) : 22 - 30.
  • 3Zhang C, Zhang J, Wei H, et al. Imbalance of NKG2D and its inhibi- tory counterparts: how does tumor escape from innate immunity? [J]. Int Immunopharmacol, 2005, 5(7-8) : 1099 - 1111.
  • 4Sutherland CL, Chalupny NJ, Schooley K, eta/. UL16-binding proteins, novel MHC class I-related proteins, bind to NKG2D and activate multiple signaling pathways in primary NK cells [ J ]. J Immunol, 2002, 168(2) : 671 -679.
  • 5Pende D, Rivera P, Marcenaro S, et al. Major histocompatibility complex class 1-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor suscepti- bility to NKG2D-dependent natural killer cell cytotexicity[ J]. Cancer Res, 2002, 62(21): 6178-6186.
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同被引文献37

  • 1吕素芳,刘峥,郭广君,蔡永萍,邱德文.大肠杆菌中表达外源重组蛋白的研究[J].科学技术与工程,2006,6(18):2872-2876. 被引量：14
  • 2郭雪艳,时永全,翟惠虹,孙力,刘理礼,韩霜,雷婷,樊代明.人核糖体蛋白S13的原核表达、纯化及多克隆抗体的制备[J].细胞与分子免疫学杂志,2007,23(4):363-366. 被引量：4
  • 3Li K, Mandai M, Hamanishi J, et al. Clinical significance of the NKG2D ligands, MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis [ J ]. Cancer Immunol lmmunother, 2009, 58(5): 641 -652,.
  • 4Wrobel P, Shojaei H, Schittek B, et al. Lysis of a broad range of epithelial tumour cells by human gamma delta T cells: involvement of NKG2D ligands and T-cell receptor-versus NKG2D-dependent recognition[J]. Stand J hnmunol, 2007, 66(2): 320-328.
  • 5Poggi A, Venturino C, Catellani S, et al. Vdeltal T lymphocytes from B-CLL patients recognize ULBP3 expressed on leukemic B cells and up-regulated by trans-retinoie aeid [ J ]. Cancer Res, 2004, 64 ( 24 ) : 9172 -9179.
  • 6Song H, Kim J, Cosman D, et al. Soluble ULBP suppresses natural killer cell activity via down-regulating NKG2D expression [ J ]. Cell Immunol, 2006, 239(1): 22-30,.
  • 7Coudert JD, Held W. The role of the NKG2D receptor for tumor immunity[J]. Semin Cancer Biol, 2006, 16(5) : 333 -343.
  • 8Pende D, Rivera P, Marcenaro S, et al. Major histocompatibility complex class l-related chain A and ULl6-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2 D-dependent natural killer cell cytotoxicity [ J ]. Cancer Res, 2002, 62(21) : 6178 -6186.
  • 9Sutherland CL, Rabinovich B, Chalupny N J, et al. ULBPs, human ligands of the NKG2D receptor, stimulate tumor immunity with enhancement by IL-15[J]. Blood, 2006, 108(4) : 1313 - 1319.
  • 10Lopez-Soto A, Quinones-Lombrana A, Lopez-Arbesu R, et al. Transcriptional regulation of ULBP1, a human ligand of the NKG2D receptor[J]. J Biol Chem, 2006, 281 (41) : 30419 -30,430.

引证文献4

二级引证文献10

细胞与分子免疫学杂志
2012年 第5期

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