【摘要】 目的制备鼠抗树鼩CD4单克隆抗体,并对其生物学特性进行鉴定。方法将重组质粒p GEX-5X-1-CD4和pET30a(+)-CD4分别转化感受态E. coli BL21和BL21(DE3),经IPTG诱导表达,并通过Glutathione Sepharose 4 Fast Flow亲和层析柱纯化重组蛋白GST-CD4和His-CD4。以His-CD4蛋白为免疫原免疫BALB/c小鼠,经杂交瘤技术筛选出能分泌抗树鼩CD4的杂交瘤细胞株,制备小鼠腹水单克隆抗体,间接ELISA法检测效价,并通过Protein A亲和层析柱纯化获得鼠抗树鼩CD4单克隆抗体,Western blot及FACS法分析其生物学特性。结果筛选出3株能稳定分泌抗树鼩CD4单克隆抗体的杂交瘤细胞株,分别命名为37D2、38E1、39F2,腹水单克隆抗体效价分别为1∶204 800、1∶204 800、1∶3 200。经HRP标记的38E1单克隆抗体可与树鼩PBMC总蛋白发生特异性结合,经PECy5. 5标记的38E1单克隆抗体能特异性识别树鼩PBMC。结论成功制备了鼠抗树鼩CD4的单克隆抗体,为进一步对树鼩作为动物模型的研究及应用奠定了基础。更多还原

【Abstract】 Objective To prepare the monoclonal antibodies(McAbs) against tree shrew CD4,and identify their biological characteristics. Methods Recombinant plasmids p GEX-5 X-1-CD4 and p ET30 a(+)-CD4 were transformed to E. coli BL21 and BL21(DE3) respectively and induced with IPTG. The expressed recombinant proteins His-CD4 and GST-CD4 were purified by Glutathione Sepharose 4 Fast Flow affinity chromatography. BALB/c mice were immunized with His-CD4 protein,and hybridoma cell lines secreting McAb against CD4 of tree shrew were screened by hybridoma technique. The prepared McAb was prepared and determined for titer by indirect ELISA,then purified by Protein A affinity chromatography and analyzed for biological characteristics by Western blot and fluorescence activated cell sorting(FACS). Results Three hybridoma cell lines stably secreting McAbs against tree shrew CD4 were screened and named as 37 D2,38 E1,39 F2,by which the titers of secreted McAbs were 1 ∶ 204 800,1 ∶ 204 800 and 1 ∶ 3 200,respectively. The McAb 38 E1 labeled with HRP showed specific binding to the total protein of PBMCs of tree shrew,while that labeled with PE-Cy5. 5 recognized the PMBCs specifically. Conclusion McAbs against CD4 of tree shrew were successfully prepared,which laid a foundation of further research and application of tree shrews as animal models.更多还原