拟步甲科昆虫不同抗冻蛋白的免疫交叉性分析

Immuno-cross-reactivity of different antifreeze proteins from Tenebrionidae insects

目的利用甲虫小胸鳖甲的抗冻蛋白MpAFP698制备抗血清,分析拟步甲科不同种类昆虫抗冻蛋白的免疫同源性。方法通过DNA疫苗初次免疫-蛋白质加强的策略免疫新西兰大白兔,先以重组质粒pcDNA3-Mpafp698作为DNA疫苗免疫接种2次,再以融合蛋白His-MpAFP698进行蛋白质加强免疫2次。采用ELISA检测MpAFP698抗体的效价,Western blot法检测抗体的特异性及不同昆虫抗冻蛋白与MpAFP698抗体的免疫交叉反应。结果兔抗血清的效价可达1:40万,Western blot结果显示抗血清分别与His-MpAFP698及GST-MpAFP698蛋白有特异性结合,与小胸鳖甲抗冻蛋白MpAFP149、MpAFPS77以及来自不同昆虫的抗冻蛋白ApAPAFP914、OcAFP4有特异性结合。结论荒漠地区不同甲虫的抗冻蛋白具有相同的抗原表位,可以发生免疫交叉反应。

Objective To prepare the antiserum against antifreeze protein, MpAFP698, from the beetle Microdera punctipennis （ M. punctipennis） and determine immunological homology of different antifreeze proteins from different insects in Tenebrionidae. Methods Two New Zealand white rabbits were immunized by DNA prime-protein boost strategy. Firstly, recombinant plasmid pcDNA3-Mpafp698 was used as DNA vaccine to inoculate two rabbits respectively for two times. Then twice boost immunization was performed using recombinant protein His-MpAFP698. ELISA and Western blotting were respectively conducted to determine the titer of MpAFP698 antibody and to detect the specificity of the antibody as well as the immunological cross-reaction of different insect antifreeze proteins with MpAFP698 antibody. Results The titer of the rabbit antiserum reached 1：400 000. Western blotting showed the specific binding of the antiserum to His-MpAFP698 protein and GST-MpAFP698 protein respectively. The rabbit antiserum also presented specific binding to antifreeze protein MpAFP149 and MpAFPS？7 from M. punctipennis, as well as Apafp014 and OcAFP4 from different species. Conclusion Antifreeze proteins from different beetles in desert region have the same epitope and thus have immunological cross-reaction.