人C型凝集素受体dectin-1单克隆抗体的制备

Preparation of monoclonal antibody of human C-type lectin receptors dectin-1

目的:克隆获得并原核表达人dectin-1基因的胞外段,以其作为抗原,制备人C型凝集素受体dectin-1的单克隆抗体。方法和结果:利用RT-PCR技术从人的外周血中克隆获得人dectin-1基因,克隆到pCDNA 3.0(+)载体中,获得pCDNA3.0(+)-dectin-1质粒。将dectin-1胞外段[dectin-1(202bp-744bp)]克隆到pET28a(+)载体,构建原核表达质粒pET28a(+)-dectin-1(202bp-744bp),转到大肠杆菌E.coli BL21(DE3)中,IPTG诱导目的片段原核表达,得到高效表达的dectin-1(202bp-744bp),镍柱亲和层析纯化后免疫BALB/c小鼠,取抗体滴度高的小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0融合,获得15株阳性杂交瘤细胞株,利用有限稀释法进行单克隆杂交瘤细胞株的筛选,获得5株单克隆细胞株。用单克隆细胞株制备小鼠腹水模型,经辛酸-硫酸铵沉淀法纯化获得dectin-1的单克隆抗体。经蛋白质印迹法验证,此单克隆抗体能有效地识别人和小鼠的dectin-1蛋白。结论:成功制备获得dectin-1蛋白的特异、高效的单克隆抗体。

Objective：To clone and prokaryoticly express the extracellular region of human C-type lectin receptor dectin-1and prepare monoclonal antibodies of human dectin-1.Methods and Results：The full-length human dectin-1mRNA was cloned from peripheral blood monocytes by using RT-PCR and inserted into the pCDNA3.0（＋）vector.The extracellular region of dectin-1[dectin-1（202bp-744bp）] was cloned and inserted into pET28a（＋）vector to get pET28a（＋）-dectin-1（202bp-744bp）vector.The recombinant pET28a-dectin-1（202bp-744bp）vector was transformed into E.coli BL21（DE3）.The expression of objective protein was high when induced with IPTG.The objective protein was purified with Ni-NTA affinity chromatograph and was used to immunize BALB/c mice.The mice which generated higher titer of antibody in the serum were sacrificed.The splenocytes were separated and fused with SP2/0cells to generate hybridoma cells.Fifteen strains of hybridoma cells were thus obtained.After further selection and cloning,5strains of monoclonal hybridoma cells secreting monoclonal antibodies against dectin-1were established.BALB/c mice was intraperitoneally injected into hybridoma cells to produce ascites.The dectin-1 monoclonal antibody was purified by standard protocol of octanoic acid-ammonium sulfate precipitation,which could specifically combine to dectin-1,no matter whether they were human dectin-1or mouse dectin-1,when used in Western blotting.Conclusion：The specific monoclonal antibodies against dectin-1were successfully obtained.