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为研究蜱类的卵黄发生及其机理,用长角血蜱Haemaphysalis longicornis卵黄蛋白(vitellin,Vn)免疫BALB/c小鼠,取免疫鼠脾细胞与骨髓瘤细胞SP2/0进行融合,经3次克隆化筛选,获得6株能稳定分泌抗Vn的单克隆抗体(McAb),即1B5,2A7,2B8,2F2,3A1和3G1。1B5,2B8和2F2亚型为IgGA;2A7亚型为IgG1;3A1和3G1亚型为IgG2a。6株McAb均具有高度特异性,效价在1∶105以上。选取效价和特异性最好的1株抗体1B5进行SDS-PAGE分析和亲和力测定,测得1B5重链和轻链的分子量分别为58kD和21kD,亲和常数为2.8993×10-6。Western免疫印迹分析发现6株单抗均与Vn的8个亚基发生免疫反应。本研究成功制备了6株抗长角血蜱Vn的单克隆抗体,为深入阐明长角血蜱卵黄发生的过程与调控机理提供了重要的工具。
In order to study the occurrence and mechanism of yolk in ticks, BALB / c mice were immunized with vitellin (Vn), and the splenocytes of immunized mice were fused with myeloma cells SP2 / 0. After three times 6 monoclonal antibodies (McAbs) that can stably secrete anti-Vn were obtained, namely 1B5, 2A7, 2B8, 2F2, 3A1 and 3G1.1B5, 2B8 and 2F2 subtypes were IgGA, 2A7 subtype was IgG1, 3A1 And 3G1 is IgG2a. All six McAbs were highly specific and their titres were above 1:105. One of the best titer and specificity antibodies, 1B5, was selected for SDS-PAGE analysis and affinity determination. The molecular weights of 1B5 heavy chain and light chain were 58kD and 21kD respectively, and the affinity constant was 2.8993 × 10-6. Western immunoblot analysis revealed that all six mAbs reacted immunoreactively with 8 subunits of Vn. In this study, we successfully prepared six monoclonal antibodies to Vn of H. pontus, which provided an important tool for further elucidating the process and regulation mechanism of yolk growth.