质粒DNA单次脾内注射制备单克隆抗体

Preparation of Monoclonal Antibody Based on Single Intrasplenic Immunization of Plasmid DNA

摘要:

探索一种新的快捷有效DNA免疫制备单克隆抗体的方法，辅助实现构建高通量无蛋白纯化体系单克隆抗体制备和筛选。分别通过“重叠PCR”和“无模板PCR”在pVAX1真核载体中分别引入IL-2信号肽、IgG kappa链信号肽构建分泌型真核表达载体，将代表抗原基因的profilin 1基因克隆到经改造带有信号肽基因的表达载体上，构建重组质粒pVAX-IL2-prof1和pVAX-Igκ-prof1，单次脾内注射重组质粒DNA免疫BALB/c小鼠。经过细胞融合、ELISA筛选，获得两株抗profilin 1的单克隆抗体。单抗亚型分别为IgM和IgG3。单次脾内质粒DNA免疫便捷有效，是制备单克隆抗体的有效方法。

Abstract:

To set up a new rapid and efficient way for the preparation of specific monoclonal antibodies (MAbs) with plasmid DNA immunization.Methods The fusion gene of IL-2 signal peptide and profilin 1 by ‘overlapping PCR' was obtained and inserted into the vector pVAX1 to construct the recombinant plasmid pVAX-IL2-prof1. Another fusion gene of IgG kappa chain signal peptide and profilin 1 by ‘no template PCR' was obtained and inserted into the vector pVAX1 to construct the recombinant plasmid pVAX-Igκ-prof1. BALB/c mice were single intrasplenic immunized with plasmid DNA. Results After cell fusion and hybridomas screening by indirect ELISA, we charactered two mAbs (1D8A2B4 and 4B12A5E3) against profilin 1. The MAbs isotype were determined as IgM and IgG3. Conclusion A single intrasplenic plasmid DNA immunization is rapid and efficient and can be used as a helpful tool for the production of specific mAb.