摘要
目的:构建并鉴定血管血友病因子裂解酶(ADAMTS13)单克隆抗体,并研究其生物学功能。方法:利用真核表达的重组血浆金属蛋白酶ADAMTS13截断型蛋白(ADAMTS13-T7)纯品免疫BALB/c小鼠,经标准单克隆抗体技术制备单克隆抗体。用ELISA方法鉴定单克隆抗体的特异性;应用免疫印迹技术确定单克隆抗体与全长ADAMTS13的识别能力;观察单克隆抗体对ADAMTS13水解v WF的影响。结果:最终获得6株抗ADAMTS13的单克隆抗体,克隆号分别为1G11、2F11、6G3、9E1、10A8、10B4。经ELISA鉴定,纯化后的单克隆抗体1G11和2F11的效价最高,与截断型ADAMTS13-T7蛋白结合能力比较,明显高于全长ADAMTS13蛋白。Western blotting结果显示,6个单克隆抗体都能与全长ADAMTS13结合,其中1G11和2F11条带最亮。功能实验表明,在变性条件下1G11和2F11能够明显抑制ADAMTS13水解v WF,并随着单克隆抗体浓度的增加而抑制作用增强。结论:成功获得针对ADAMTS13的单克隆抗体,其中两株为抑制性功能抗体。
Objective:To construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13),and to study their biological function.Methods:BALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein(ADAMTS13-T7).Murine anti-human ADAMTS13 monoclonal antibodies(McAbs) were constructed by standard hybridoma technology and identified by ELISA.The recognition of McAbs with full4 ength recombinant ADAMTS13 protein was identified by Western blot.In function assay,the influence of McAbs on the proteolysis of vWF by ADAMTS13 was observed.Results:A group of 6 murine anti-ADAMTS13 McAbs was obtained with the clone number 1G11,2F11,6G3,9E1,10A8 and 10B4.In ELISA,the highest titers of1G11 and 2F11 were observed,both of which showed a higher affinity to ADAMTS13-T7 than full-length ADAMTS13.The Western blot demonstrated that the 6 McAbs all could recognize ADAMTS13,among which 1G11 and 2F11 showed stronger reaction with ADAMTS13.In addition,under the denatured conditions,1G11 and 2F11 could inhibit hydrolysis of vWF by ADAMTS13,and that was stronger with the increasing of McAbs concentration.Conclusion:McAbs against ADAMTS13 have been gained,two of which are inhibitory antibodies.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2016年第4期1104-1109,共6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)
