EBV编码潜伏膜蛋白2A抗原表位的串联表达及其免疫原性分析

Preparation and immunogenicity characteristics analysis of a epitope fusion protein of Epstein-Bar virus encoded latent membrane protein 2A

目的:制备具有免疫原性的EB病毒潜伏膜蛋白2A(latent membrane protein 2A,LMP2A)的表位串联蛋白并分析其免疫学特性。方法:用DNAstar软件分析LMP2A的抗原表位,将预测的免疫原性较强的两个表位通过基因合成串联在一起,克隆到原核表达载体pET28a中,经大肠杆菌BL21表达并纯化。制备的含LMP2A表位重组蛋白经SDS-PAGE、Western blot鉴定后,免疫小鼠制备多克隆抗体,以ELISA检测抗体的效价,免疫组织化学法检测该抗体对天然LMP2A的特异性。结果:通过原核表达与纯化,获得高纯度的表位融合蛋白,经小鼠免疫并制备效价高且特异性的小鼠抗LMP2A的多克隆抗体,该抗体可用于ELISA和免疫组织化学分析。结论:本研究制备的表位融合蛋白,具有天然抗原的免疫原性,可制备能特异性识别天然LMP2A分子的多克隆抗体,为利用表位融合蛋白筛选全人源基因工程抗体奠定了基础。

Objective：To prepare a immunogenic epitope tandem of latent membrane protein 2A （LMP2A） of Epstein-Barr virus and analyze its characteristics of immunology. Methods：LMP2A epitopes were analyzed by using DNAstar software,and two immunogenic epitopes were selected due to stronger immunogenicity. The genes of these two epitopes were integrated through gene synthesis, and then cloned into prokaryotie expression vector pET28a. The recombinant epitopes were expressed and purified by E.coli BL21,and then the protein was utilized to immunize mouse to prepare anti-epitope fusion protein polyclonal antibody after the analysis of SDS-PAGE and Western blot assay. The titer of the antibody was assessed by ELISA and the immunobistochemical experiment was performed to test the specificity of the polyclonal antibody for natural LAMP2A. Results：Through prokaryotic expression and purification, high purity fusion protein was obtained. By mouse immunization, anti-LMP2A polyclonal antibody of high titer and specificity was prepared, which can be applied to ELISA and immunohistochemical analysis. Conclusion：An epitope fusion protein,which shares the natural antigen immunogenicity,can be used to prepare the polyclonal antibody specific for LMP2A. This study has laid a solid foundation for screening whole humanized genetic engineering antibody by epitope fusion protein.