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利用hTM表达细胞株制备抗人血栓调节蛋白单克隆抗体
引用本文:郭紫芬,何淑雅,朱炳阳,李斌元,廖端芳.利用hTM表达细胞株制备抗人血栓调节蛋白单克隆抗体[J].生物化学与生物物理进展,2009,36(4):441-447.
作者姓名:郭紫芬  何淑雅  朱炳阳  李斌元  廖端芳
作者单位:1. 南华大学药物药理研究所,衡阳,421001
2. 南华大学生物化学教研室,衡阳,421001
基金项目:国家自然科学基金资助项目(30770868)和湖南省重点学科建设项目.
摘    要:为了研制抗人血栓调节蛋白(hTM)的单克隆抗体(mAb),给经CHO细胞免疫的BALB/c小鼠腹腔注射环磷酰胺诱导其对CHO和CHO-TM5细胞的共同抗原表位产生免疫耐受,再用高效表达hTM的CHO-TM5常规免疫小鼠.细胞融合后,ELISA筛选分泌抗hTM的特异性mAb阳性克隆,并将其腹腔注射BALB/c小鼠诱生腹水.腹水中的mAb经亲和层析纯化后,采用ELISA、流式细胞术、免疫组织化学染色法及Western blotting对其进行特异性鉴定.结果显示,共获得了5株阳性克隆,其中2F7可稳定分泌IgG1亚型的mAb,腹水抗体效价为1×10-6,含量为19.56 g/L.2F7在体内与正常组织的交叉反应极少,对HUVEC、CHO-TM5有特异性结合,并可识别细胞裂解液还原条件下分子质量为105 ku左右的蛋白质多肽.2F7的解离常数Kd约为1.22×10-9 mol/L.实验结果表明,通过采用新的技术路线,直接应用hTM表达细胞株免疫小鼠,成功制备了具有高度特异性与高亲和力的抗hTM mAb,为其他来源困难的蛋白质mAb制备提供了可借鉴的技术方案,同时2F7的研究鉴定为进一步应用抗hTM mAb进行hTM生物学功能及临床意义研究提供了新的物质基础.

关 键 词:免疫耐受  环磷酰胺  血栓调节蛋白  单克隆抗体
收稿时间:2008/7/19 0:00:00
修稿时间:2/6/2009 12:00:00 AM

Preparation of Anti-hTM Monoclonal Antibody by Using hTM Expression Cell Line
GUO Zi-Fen,HE Shu-Y,ZHU Bing-Yang,LI Bin-Yuan and LIAO Duan-Fang.Preparation of Anti-hTM Monoclonal Antibody by Using hTM Expression Cell Line[J].Progress In Biochemistry and Biophysics,2009,36(4):441-447.
Authors:GUO Zi-Fen  HE Shu-Y  ZHU Bing-Yang  LI Bin-Yuan and LIAO Duan-Fang
Institution:Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China;Department of Biochemistry, University of South China, Hengyang 421001, China;Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China;Department of Biochemistry, University of South China, Hengyang 421001, China;Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China
Abstract:To produce monoclonal antibody (mAb) specifically against human thrombomodulin (hTM), an immune-tolerizing procedure was employed to generate monoclonal antibodies specific to hTM. Female BALB/c mice were first immunized with CHO cells following at 10 min, 24 h, 48 h by intraperitoneal injection of different doses of cyclophosphamide (CP) 2 times at an interval of 2 weeks, thereby tolerizing the mice to common epitopes shared between CHO and CHO-TM5 cells. Subsequently the selected mice with the lowest titer of serum polyclonal antibody by cellular enzyme-linked immunoabsorbent assay (CELISA) were immunized with CHO-TM5 cells, which have stable high level expression of hTM, to produce antibodies specific to hTM 3 times at an interval of 2 weeks. On the third day after the third immunization, mouse with the highest titer of serum polyclonal antibody was sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with CELISA. To improve probability to obtain specific mAb, CELISA was applied twice. The first CELISA was done with polyethylene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but having CHO cells monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5+CHO- hybridoma cells. BALB/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded mAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Detection of CELISA showed that 100 mg/kg dose of CP could tolerize the mouse to common epitopes shared between CHO and CHO-TM5 cells. And CELISA also discovered that all hybridomas positive for CHO-TM5 cells were negative for CHO cells. Five lines of positive hybridoma cells had been obtained altogether and 2F7 was selected randomly for next investigation. The Ig subclass of the mAb 2F7 was IgG1 and the titer of ascitic mAb was 1×10-6. Furthermore, the content of ascitic mAb was 19.56 g/L and chromosome numbers is 98. Flow cytometry, CELISA and Western blotting assays demonstrated that mAb 2F7 could specifically recognize hTM expressed on CHO-TM5 and human umbilical vascular endothelial cells (HUVEC). Meanwhile, the tissue specificity of mAb 2F7 was also identified by immunohistochemical ABC staining. On the other hand, Western blotting assays indicated that mAb 2F7 could recognize the antigen protein with 105 ku molecular mass under reduction condition. Moreover, the dissociation constant of mAb 2F7, 1.22×10-9 mol/L, indicated the affinity higher than some others. The results suggest that the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms. mAb 2F7 can specifically recognize the natural hTM expressed mainly on vascular endothelial cells, which will potentially useful for investigating the functions and clinic values of hTM.
Keywords:immune tolerance  cyclophosphamide  thrombomodulin  monoclonal antibodies
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