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[Immuno-cross-reactivity of different antifreeze proteins from Tenebrionidae insects]
Jieqiong Li1, Xiaojuan Hou, Yan Wang
1College of Life Science and Technology, Xinjiang University, Urumqi, China.
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 MpAFPS77 from M.punctipennis, as well as Apafp914 and OcAFP4 from different species.
Conclusion:
Antifreeze proteins from different beetles in desert region have the same epitope and thus have immunological cross-reaction.
