C型凝集素受体Dectin-2和Dectin-3的单克隆抗体制备  

Preparation of monoclonal antibodies against C-type lectin receptors Dectin-2 and Dectin-3

作　　者：关建洪[1] 朱乐乐[1] 林欣[1] 贾鑫明[1] 

机构地区：[1]同济大学医学院免疫学教研室,上海200092

出　　处：《同济大学学报（医学版）》2014年第2期1-7,共7页Journal of Tongji University(Medical Science)

基　　金：上海市基础研究重点基金(12JC1408400)

摘　　要：目的克隆人C型凝集素受体dectin-2和dectin-3胞外区基因并进行原核表达及制备它们的单克隆抗体。方法利用PCR扩增编码人dectin-2和dectin-3胞外区的基因序列,分别克隆到原核表达载体pET28a(+)中,IPTG诱导基因的原核表达;纯化后免疫Balb/c小鼠,融合并筛选获得克隆化的杂交瘤细胞株,制备Dectin-2和Dectin-3的单克隆抗体;利用ELISA、FACS和Western印迹法检测抗体的效价和特异性;检测Dectin-2和Dectin-3的单克隆抗体是否能够特异性阻断相应受体的功能。结果成功构建原核表达质粒pET28a(+)-Dectin-2和pET28a(+)-Dectin-3,并在E.coli BL21(DE3)中诱导Dectin-2和Dectin-3胞外区的高效表达。纯化目的蛋白后免疫Balb/c小鼠,融合获得8株Dectin-2和12株Dectin-3的阳性杂交瘤细胞。进一步克隆化筛选获得7株Dectin-2和8株Dectin-3的单克隆细胞株,并且其分泌的抗体能够特异结合Dectin-2和Dectin-3胞外区的原核表达蛋白。利用单克隆细胞株制备小鼠腹水,纯化获得Dectin-2和Dectin-3的单克隆抗体。单克隆细胞株D2-7和D3-2产生的抗体能够特异结合RAW中表达的人Dectin-2和Dectin-3蛋白及小鼠骨髓来源巨噬细胞中Dectin-2和Dectin-3蛋白。抗体功能实验显示:单克隆细胞株D2-7和D2-10产生的抗体能够阻断Dectin-2受体介导白念珠菌菌丝态刺激引起的NF-κB激活,而单克隆细胞株D3-1和D3-2产生的抗体能够阻断Dectin-3受体介导白念珠菌菌丝态刺激引起的NF-κB激活,并且具有剂量依赖性及高度特异性。结论成功获得高纯度的Dectin-2和Dectin-3胞外区蛋白,并分别获得了特异、高效价的单克隆抗体。Objective To prepare monoclonal antibodies against human C-type lectin receptors Dectin-2 and Dectin-3. Methods The prokaryotic expression plasmids pET28a（ ＋）-Dectin-2 and pET28a（ ＋）-Dectin-3 were constructed by using molecular cloning technique,and then transfected to E. coli BL21（ DE3） to induce protein expression with IPTG. The purified proteins were used to immunize Balb / c mice to obtain monoclonal hybridoma cells and prepare monoclonal antibodies against Dectin-2 and Dectin-3,respectively. The titer and specificity of prepared antibodies were detected by ELISA, FACS and Western blotting. The functional blockage of Dectin-2 and Dectin-3 by prepared monoclonal antibodies was verified. Results The extracellular region of Dectin-2 and Dectin-3 were cloned and expressed in E. coli BL21（ DE3） with high efficiency. Eight and 12 strains of hybridoma cells for Dectin-2 and Dectin-3 were obtained from Balb / c mice immunized with the purified proteins. After further selection and cloning,7 strains of monoclonal hybridoma cells secreting monoclonal antibodies against Dectin-2,and 8 strains for Dectin-3 were established. These monoclonal hybridoma cells were injected into celiac cavity of mice and monoclonal antibodies against Dectin-2 or Dectin-3 were harvested from collected ascites and purified. The antibodies produced from strains D2-7 and D3-2 were able to specifically combine with Dectin-2 and Dectin-3 from RAW cells or from mouse bone marrow-derived macrophages. The antibodies produced from strains D2-7 and D2-10 completely inhibited the NF-κB activation mediated by Dectin-2 in response to C. albicans hyphae stimulation; the antibodies produced from strains D3-1 and D3-2 specifically inhibited the NF-κB activation mediated by Dectin-3 in response to C. albicans hyphae in a dose-dependent manner. Conclusion The purified monoclonal antibodies against Dectin-2 and Dectin-3 have been successfully prepared with high titer and specificity.

关 键 词：C型凝集素受体 Dectin-2蛋白 Dectin-3蛋白 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]