Trim22 B细胞表位预测及多克隆抗体制备与鉴定

Prediction of the B cell epitope of Trim22 and preparation of polyclonal antibodies

目的:预测人Trim22的B细胞抗原表位,制备并鉴定其多克隆抗体。方法:利用生物信息学软件分析Trim22的氨基酸序列,确定抗原表位并人工合成多肽。将合成肽与牛血清白蛋白(BSA)偶联,免疫大白兔制备抗Trim22的抗体。经饱和硫酸铵沉淀法纯化抗体,并对抗体进行ELISA、Western blot鉴定。结果:获得抗Trim22的多克隆抗体,抗体效价为1∶16 000,该抗体能特异性地识别包含(382～397)区段的Trim22缺失突变体,同时该抗体不能识别Trim22旁系同源分子Trim5α、Trim6和Trim34α。结论:采用人工合成多肽作为半抗原制备出抗Trim22的多克隆抗体,对研究内源性Trim22与HIV-1衣壳蛋白的相互作用机制具有重要价值。

Objective：To predict the B cell epitope of human Trim22 so as to preparte and identify polyclonal antibody induced by synthesized peptides.Methods：B cell dominant epitopes of Trim22 were predicted by the method of secondary structure,hydrophilicity,antigenicity,accessibility and flexibility.The synthesized peptides were cross-linked with bovine serum album（BSA）,which was used to immunize rabbits.The polyclonal antibodies were purified by saturated ammonium sulfate,and then they were characterized by ELISA and Western blot.Results：The polyclonal antibodies against Trim22 were prepared successfully,with the titer of 1∶16 000.Western blot identified that the antibodies could recognize Trim22 truncation mutant containing the sequence of 382-397,whereas the antibodies could not recognize the Trim22 paralogous molecules,such as Trim5α,Trim6 and Trim34α.Conclusion：The synthesized peptides for Trim22 can be used to immunize rabbits to generate the polyclonal antibodies successfully.It will be propitious for the intensive study of the interactions between endogenous Trim22 and HIV-1 Gag protein.