酵母表面展示多肽片段分析Δ42PD1的免疫原性

发布时间：2018-07-22 20:04

【摘要】：目的:分析Δ42PD1胞外区的免疫原性。方法:将Δ42PD1胞外区编码基因分为6个片段,分别用PCR的方法进行扩增,并克隆至酵母表面展示质粒p CTCON2。分别将重组酵母表达质粒转化酵母细胞,涂布SDCAA平板,挑取单细胞克隆于SGCAA培养基中诱导目的基因的表达。通过肌肉注射Δ42PD1真核表达质粒加局部电穿孔的方法免疫BALB/c小鼠,制备鼠抗人Δ42PD1免疫血清。用流式细胞术的方法分析免疫血清与酵母表面展示的Δ42PD1多肽片段的结合。结果:PCR扩增的Δ42PD1的6个片段的编码序列全长均110 bp,核酸序列分析显示克隆的目的基因与Gen Bank中已经登记的PD1基因序列100%同源;流式细胞术的结果发现酵母表面展示的Δ42PD1的F3和F2片段可与Δ42PD1的免疫血清发生明显结合。结论:Δ42PD1的F3和F2片段是该分子的免疫优势区域,为制备Δ42PD1的单克隆抗体及表位筛选奠定了基础。
[Abstract]:Objective: to analyze the immunogenicity of 螖 42 PD1 extracellular domain. Methods: the encoding gene of 螖 42PD1 extracellular domain was divided into six fragments and amplified by PCR and cloned into yeast surface display plasmid pCTCON2. The recombinant yeast expression plasmids were transformed into yeast cells and coated with SDCAA plate. The single cells were selected and cloned into SGCAA medium to induce the expression of the target gene. BALB / c mice were immunized by intramuscular injection of 螖 42PD1 eukaryotic expression plasmid and local electroporation to prepare mouse anti-human 螖 42PD1 immune serum. The binding of immune serum to 螖 42PD1 polypeptide fragment displayed on yeast surface was analyzed by flow cytometry. Results the six fragments of 螖 42PD1 amplified by 10% PCR were all 110bp in length. The nucleic acid sequence analysis showed that the cloned target gene was 100% homologous to the PD1 gene previously registered in GenBank. The results of flow cytometry showed that the F3 and F2 fragments of 螖 42PD1 displayed on the surface of yeast could bind to the immune serum of 螖 42PD1. Conclusion: the F _ 3 and F _ 2 fragments of 螖 42PD1 are the dominant immunological regions of 螖 42PD1, which lays a foundation for the preparation of monoclonal antibodies against 螖 42PD1 and the screening of epitopes.
【作者单位】：深圳市第三人民医院;
【基金】：国家自然科学基金(31500750) 广东省自然科学基金(2016A030313030) 深圳市科技创新基金(JCYJ20140411111718162,JCYJ20150402111430650)资助项目
【分类号】：R392

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