抗ADAMTS13单克隆抗体杂交瘤细胞株的建立  

Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies against ADAMTS13

作　　者：凌婧[1] 马珍妮[2] 殷杰[2] 沈飞[2] 谢丽倩[2] 赵云霄[2] 阮长耿[2] 

机构地区：[1]苏州大学附属儿童医院血液科,江苏苏州215000 [2]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,江苏苏州215000

出　　处：《中国实验血液学杂志》2017年第4期1123-1129,共7页Journal of Experimental Hematology

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)

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。Objective： To establish the hybridoma cell lines secreting anti-ADAMTS13-T7 monoclonal antibodies（ MA6） and to construct the MAb directed against different domains of ADAMTS13-T7. Methods： The trucated type protein ADAMTS13 of eukaryote-expressed recombinant ADAMTS13-T7 was constructed and was purified by using NiNTA agarose. Then the purified ADAMTS13 was used to immunize the BALB/c mice; the antiserum titer of ADAMTS13 protein of immunized mice was deteceted by ELISA. The spleen was taken from mice for construcing the single cell suspension,then the single cell suspension was mixed with myeloma cells SP2/0 at 10∶ 1 ratio for cell fusion,and the elution culture of hybridoma cells was carried out; after 2 weeks,the wells in which clones were well grown were selected for detection,then the positive clonal wells were expansively cultured and the detection again was performed. Results： The purified ADAMTS13-T7 protein was gained. The ELISA showed that the antiserum titer in mice immumized by ADAMTS13-T7 protein was 1 ∶ 20000. The results of fusion culture by hybridoma techmique showed that 30 hyridoma cell lines secreting MAb against recombinant ADAMTS13 were established. Conclusion： The hybridoma cell lines secreting MAb against recombinant ADAMTS13 have been successfully established by fusion culture,which provide more powerful tools for further screening the MAb with certain functional activity and studying the structure and function of ADAMTS13.

关 键 词：ADAMTS13 真核表达 杂交瘤 单克隆抗体 

分 类 号：R364.15[医药卫生—病理学] R554.6[医药卫生—基础医学]