异色瓢虫卵黄蛋白单克隆抗体的制备及鉴定  

Preparation and identification of monoclonal antibodies to the vitellin in Harmonia axyridis( Coleoptera: Coccinellidae)

作　　者：马卓[1,2] 刘廷辉[1] 陈洁[1,3] 梁超[1] 曹美琳[1] 何运转[1] 

机构地区：[1]河北农业大学植物保护学院,河北保定071000 [2]中国农业大学农学与生物技术学院,北京100094 [3]邯郸市农业科学院,河北邯郸056001

出　　处：《昆虫学报》2015年第11期1186-1193,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31272095);河北省中药材产业技术体系资助项目

摘　　要：【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。[Aim ] In order to accurately track the synthesis,transport pathway and absorption of vitellogenin（ Vg） in Harmonia axyridis（ Pallas） as well as the accumulation and distribution of vitellin（ Vn） in the oocytes,the monoclonal antibody（ Mc Ab） against Vn of H. axyridis was prepared.[Methods]The hybridoma cell lines secreting Mc Ab against Vn of H. axyridis were produced by using hybridoma techniques,based on the cells from BALB / c mouse which were immunized with the Vn.[Results]We produced four monoclonal antibodies to H. axyridis soluble yolk proteins,i. e.,5E2,5E11,1E9 and 5H8. Further identification indicated that 1E9,5E11 and 5E2 were the isotype Ig G1,and 5H8 was the isotype Ig M. Western blot analysis showed that the four antibodies had high specificity and affinity to the Vn. However,they showed no immunological reaction with the male haemolymph. 5E2 and 1E9 had specific immunological reaction with the four subunits of the Vn. Finally,we selected 5E2 whose titer was the highest before ascites preparation to prepare the monoclonal antibodies. SDS-PAGE indicated that the molecular masses of 5E2 heavy chain and light chain were 50 and 27 k D,respectively,and its titer was 1 ∶ 81 000. [Conclusion]The successful preparation and identification of H. axyridis Mc Ab may establish an essential tool for further building the ELISA method for assaying its dynamics.

关 键 词：异色瓢虫 卵黄蛋白 单克隆抗体 亚型 亚基鉴定 

分 类 号：Q966[生物学—昆虫学]