抗ADAMTS13单克隆抗体杂交瘤细胞株的建立

发布时间：2018-07-29 15:31

【摘要】：目的:建立分泌抗ADAMTS13单克隆抗体的杂交瘤细胞株,制备针对ADAMTS13不同结构域的单克隆抗体。方法:利用真核表达的重组血浆金属蛋白酶ADAMTS13截短型蛋白(ADAMTS13-T7)纯品免疫BALB/c小鼠。用ELISA方法测定免疫小鼠与ADAMTS13-T7蛋白的抗血清效价。取小鼠脾脏,制成单细胞悬液与SP2/0骨髓瘤细胞以10∶1的比例混合,进行细胞的融合,并将杂交瘤细胞稀释培养。2周后选生长良好的克隆孔检测,阳性克隆孔扩大培养并再次检测。结果:获得真核表达截短型血管性血友病因子裂解蛋白酶ADAMTS13-T7蛋白纯品。利用真核表达的截短型ADAMTS13-T7蛋白免疫BALB/c雌性小鼠,ELISA检测免疫后抗血清效价约为1∶20 000。利用杂交瘤技术通过融合,获得多株分泌抗重组ADAM TS13抗体的杂交瘤单克隆细胞株。将其中的30株转入液氮中冻存,以备进一步分选及功能研究。结论:融合获得多株分泌抗重组ADAMTS13抗体的杂交瘤单克隆细胞株,为进一步筛选出具有一定功能活性的单克隆抗体及研究ADAMTS13结构和功能提供更多的有力工具。
[Abstract]:Aim: to establish hybridoma cell lines secreting monoclonal antibodies against ADAMTS13 and to prepare monoclonal antibodies against different domains of ADAMTS13. Methods: BALB/c mice were immunized with recombinant plasma metalloproteinase ADAMTS13 truncated protein (ADAMTS13-T7). The antiserum titers of immunized mice and ADAMTS13-T7 protein were determined by ELISA method. The spleen of mice was taken and the monocyte suspension was mixed with SP2/0 myeloma cells at 10:1 to fuse the cells. The hybridoma cells were diluted and cultured for 2 weeks. The positive clones were expanded and tested again. Results: eukaryotic expression of truncated von Willebrand factor lysase ADAMTS13-T7 protein was obtained. The antiserum titer of BALB/c female mice immunized with truncated ADAMTS13-T7 protein was about 1:20 000 by Elisa. A number of hybridoma monoclonal cell lines secreting anti-recombinant ADAM TS13 antibody were obtained by fusion with hybridoma technique. 30 of them were frozen in liquid nitrogen for further sorting and functional study. Conclusion: a number of hybridoma cell lines secreting anti-recombinant ADAMTS13 antibodies were obtained by fusion, which provide more powerful tools for further screening monoclonal antibodies with certain functional activities and for studying the structure and function of ADAMTS13.
【作者单位】：苏州大学附属儿童医院血液科;苏州大学附属第一医院江苏省血液研究所卫生部血栓与止血重点实验室;
【基金】：国家自然科学基金(81500107) 江苏省基础研究计划(自然科学基金)-青年基金项目(BK20140285)
【分类号】：R55

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