长角血蜱卵黄蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibodies to the vitellin from the hard tick Haemaphysalis longicornis Neumann(Arachnida:Ixodidae)

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。

This study aims at tick vitellogenesis and its mechanism. Six hybridoma cell lines secreting monoclonal antibody （McAb） against vitellin （Vn） of the hard tick Haemaphysalis longicornis were produced by fusing myeloma cells （SP2/0） with spleen ceils, both from BALB/c mouse which were immunized with the Vn. They were named as 1B5, 2A7, 2B8, 2F2, 3A1 and 3G1, respectively. Further identification indicated that 1B5, 2B8 and 2F2 were of the isotype IgGA, 2A7 was of the isotype IgG1, while 3A1 and 3G1 were of the isotype IgG2a. All the six antibodies had high specificity and affinity, and their titers were over 10-5 . SDS-PAGE and affinity analysis of 1B5, whose specificity and titer were the highest, indicated that the molecular masses of its heavy chain and light chain were 58 kD and 21 kD, respectively, and the affinity constant was 2. 8993 ×10-6. Western blot analysis showed that the six antibodies had specific immunological reaction with the eight subunits of the Vn. The six MeAbs against the Vn of H. longicorns prepared and identified successfully in this study may provide an important tool for further elucidating vitellogenesis and its regulation mechanism in H. longicorns.