P2X7受体短肽单克隆抗体的制备及鉴定

生物技术通报 ›› 2012, Vol. 0 ›› Issue (12): 167-172.

P2X7受体短肽单克隆抗体的制备及鉴定

谭超^{1, 2}, 韩莉¹, 神谢静¹

1 三峡大学医学院，宜昌 443002 ；2 三峡大学第一临床医学院宜昌市中心人民医院检验科，宜昌 443002

收稿日期:2012-07-06 修回日期:2013-01-25 出版日期:2012-12-26 发布日期:2013-02-06
作者简介:谭超, 男, 硕士研究生, 主管技师, 研究方向:分子免疫学;E-maiyczxyytanchao@sina.com
基金资助:
: 湖北省自然科学基金资助项目（2009CDZ024）, 三峡大学研究生科研创新基金项目（2011CX059）

The Preparation and Identification of P2X7 Short Peptide ReceptorMonoclonal Antibody

Tan Chao^1,2, Han Li¹, Shen Xiejing¹

1 Medical Science College of China Three Gorges University，Yichang 443002 ；2Department of Internal Medicine Yichang Central People’sHospital Laboratory Medicine，the First Clinical Medical College of China Three Gorges University，Yichang 443002

Received:2012-07-06 Revised:2013-01-25 Published:2012-12-26 Online:2013-02-06

摘要/Abstract

摘要： 旨在制备与鉴定鼠抗P2X7 受体的蛋白单克隆抗体。以人P2X7 受体的胞外段制备短肽作为抗原，皮下注射免疫Balb/c 小鼠。分离小鼠脾脏B 淋巴细胞与骨髓瘤细胞融合并培养，挑选阳性杂交瘤细胞，扩大培养，制备和鉴定P2X7 其生物学效应。结果显示，获得1 株稳定分泌抗人P2X7 受体的单克隆抗体的杂交瘤细胞株，所分泌的单抗类型重链为IgG1，轻链为κ ；该株杂交瘤细胞腹水效价为1∶6.4×10⁴；传30 代及液氮中保存6 个月，抗体效价稳定；Western blotting 检测证明该单抗与人细胞表面的P2X7 受体蛋白特异地结合。所制备的抗人P2X7 受体的单克隆抗体具有高度的特异性及稳定性，为针对P2X7 受体为靶点的抗体药物的开发应用、疾病的辅助诊断奠定了基础。

关键词: P2X7, 受体, 短肽, 单克隆抗体

Abstract: The aim was to prepare and identify the monoclonal antibodies of the P2X7 receptor. Our method was to use the extracellularfragment of human beings’ P2X7 receptor to produce the short peptide as an antigen to immunize mouse for three times. Then B lymphocyteswere isolated from the mouse spleen, and then mixed with myeloma cells and cultured. The positive hybridoma clones were selected and widelycultured to prepare and identify the biological effects of P2X7. The supernatant was collected and purified by HiTrap protein G affinity column.The results were as follows. Firstly, a stable hybridoma cell line secreting mAb against human P2X7 receptor was obtained, and the heavy andlight chains of the secreted mAb were IgG1 and κ respectively. The titers of the ascites was 1∶6.4×104, which was remained stable aftersubculture of hybridoma cells for 30 passages or storage at liquid nitrogen for 6 months. Secondly, the western blotting test showed that the mAbcould specifically bind to P2X7 receptor. The conclusion was that the prepared mAb against P2X7 receptor showed high specificity and stability,which laid a foundation for the development of the antibody drug aimed at P2X7 receptor as a target antigen.

谭超, 韩莉, 神谢静. P2X7受体短肽单克隆抗体的制备及鉴定[J]. 生物技术通报, 2012, 0(12): 167-172.

Tan Chao, Han Li, Shen Xiejing. The Preparation and Identification of P2X7 Short Peptide ReceptorMonoclonal Antibody[J]. Biotechnology Bulletin, 2012, 0(12): 167-172.

参考文献

[1] Miller CM, Boulter NR, Fuller SJ, et al, The role of the P2XT receptorin infectious diseases. PLoS Pathog, 2011, 7(11)：e1002212.
[2] Guerrero-Alba R, Valdez-Morales E, Juárez EH, et al. Two suraminbinding sites are present in guinea pig but only one in murine nativeP2X myenteric receptors. Eur J Pharmacol, 2010, 626(2-3)：179-185.
[3] Samways DS, Khakh BS, Dutertre S, et al. Preferential use ofunobstructed lateral portals as the access route to the pore of humanATP-gated ion channels(P2X receptors). Proc Natl Acad SciUSA, 2011, 108(33)：13800-13805.
[4] Burnstock G, Fredholm BB, Verkhratsky A. Adenosine and ATP receptorsin the brain. Curr Top Med Chem, 2011, 11(8)：973-1011.
[5] Lenertz LY, Gavala ML, Zhu Y, et al. Transcriptional control mechanismsassociated with the nucleotide receptor P2X7, a critical regulatorof immunologic, osteogenic, and neurologic functions. ImmunolRes, 2011, 50(1)：22-38.
[6] Deli T, Csernoch L. Extracellular ATP and cancer: an overview withspecial reference to P2 purinergic receptors. Pathol Oncol Res, 2008,14(3)：219-231.
[7] Li X, Qi X, Zhou L, et al. P2X(7)receptor expression is decreasedin epithelial cancer cells of ectodermal, uro-genital sinus, and distalparamesonephric duct origin. Purinergic Signal, 2009, 5(3)：351-368.
[8] Shih H, Lee B, Lee RJ, et al. The aging heart and post-infarction leftventricular remodeling. J Am Coll Cardiol, 2011, 57(1)：9-17.
[9] Delarasse C, Gonnord P, Galante M, et al. Neural progenitor celldeath is induced by extracellular ATP via ligation of P2X7 receptor.J Neurochem, 2009, 109(3)：846-857.
[10] Jursik C, Sluyter R, Georgiou JG, et al. A quantitative method forroutine measurement of cell surface P2X7 receptor function inleucocyte subsets by two-colour time-resolved flow cytometry. JImmunol Methods, 2007, 325(1-2)：67-77.
[11] Burnstock G, Verkhratsky A. Long-term(trophic)purinergicsignalling: purinoceptors control cell proliferation, differentiationand death. Cell Death Dis, 2010, e9.
[12] Sun C, Chu J, Singh S, et al. Identification and characterization of anovel variant of the human P2X(7)receptor resulting in gain offunction. Purinergic Signal, 2010, 6(1)：31-45.
[13] Im Hof M, Williamson L, Summerfield A, et al. Effect of syntheticagonists of toll-like receptor 9 on canine lymphocyte proliferationand cytokine production in vitro. Vet Immunol Immunop, 2008, 124(1-2)：120 - 131.
[14] Wang M, Tan J, Wang Y, et al. IL-18 binding protein- expressingmesenchymal stem cells improve myocardial protection after ischemiaor infarction. Proc Natl Acad Sci USA, 2009, 106(41)：17499-17504.
[15] Braeuninger S, Kleinschnitz C, Stoll G. Interleukin-18 does notinfluence infarct volume or functional outcome in the early stageafter transient focal brain ischemia in mice. Exp Transl Stroke Med,2010, 2: 1.
[16] Iannello A, Samarani S, Debbeche O, et al. Potential role ofinterleukin-18 in the immunopathogenesis of AIDS: involvementin fratricidal killing of NK cells. J Virol, 2009, 83(12)：5999-6010.
[17] Friedle SA, Brautigam VM, Nikodemova M, et al. The P2X7-Egr pathway regulates nucleotide -dependent inflammatory geneexpression in microglia. Glia, 2011, 59(1)：1-13.
[18] Gorodeski GI．P2X7-mediated chemoprevention of epithelial cancers.Expert Opin Ther Targets, 2009, 13(11)：1313-1332.