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【目的】为了能准确地追踪异色瓢虫Harmonia axyridis(Pallas)卵黄原蛋白(vitellogenin,Vg)的合成、转运途径和吸收方式,以及卵黄蛋白(vitellin,Vn)在卵母细胞内的积累及分布情况,本研究对异色瓢虫的Vn进行了单克隆抗体(monoclonal antibody,Mc Ab)的制备。【方法】以异色瓢虫Vn免疫BLAB/C小鼠,应用杂交瘤技术,经过3次亚克隆筛选,制备能稳定分泌抗Vn的单克隆抗体。【结果】实验获得4株能够稳定分泌抗异色瓢虫Vn的单克隆抗体,即5E2,5E11,1E9和5H8。其中1E9,5E11和5E2亚型均为Ig G1,5H8亚型为Ig M。Western blot免疫印迹分析显示,4株单克隆抗体可以特异性地识别Vn,而与雄虫血淋巴无反应。其中,5E2和1E9可以与异色瓢虫抗原的4个亚基发生较强的免疫反应,结合腹水制备前上清效价检测结果最终选取5E2制备单克隆抗体。5E2单克隆抗体的效价为1∶81 000,SDS-PAGE分析显示5E2重链和轻链的分子量分别为50和27 k D。【结论】本实验成功制备出一株能够稳定分泌抗异色瓢虫Vn的单克隆抗体,为建立酶联免疫吸附试验(ELISA)方法测定其动态变化奠定了基础。
【Objective】 In order to accurately trace the synthesis, transport and absorption of vitellogenin (Vg) of Harmonia axyridis (Pallas) and the accumulation of vitellin (Vn) in oocytes and In this study, monoclonal antibodies (McAbs) were prepared for Vn of C. heterochina. 【Method】 BLAB / C mice were immunized with Vn of C. heterophyllus and monoclonal antibodies against Vn were prepared by subcloning three times with hybridoma technique. 【Result】 Four monoclonal antibodies against Vn of Isochrysis were obtained stably, that is, 5E2, 5E11, 1E9 and 5H8. Which 1E9, 5E11 and 5E2 subtypes are Ig G1, 5H8 subtype is Ig M. Western blot analysis showed that four monoclonal antibodies could specifically recognize Vn, but no reaction with male hemolymph. Among them, 5E2 and 1E9 could react strongly with the 4 subunits of the heterochromous ladybug antigen, and the final selection of 5E2 in combination with the test results of the ascites before preparation of the supernatant was used to prepare the monoclonal antibody. The titer of the 5E2 monoclonal antibody was 1:81 000 and SDS-PAGE analysis showed that the molecular weights of the 5E2 heavy and light chains were 50 and 27 kD, respectively. 【Conclusion】 This experiment successfully prepared a monoclonal antibody that can stably secrete Vn against C. elegans, which lays the foundation for the establishment of the enzyme-linked immunosorbent assay (ELISA) method for the determination of its dynamic changes.