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酵母表面展示多肽片段分析Δ42PD1的免疫原性

Analysis of immunogenicity of Δ42PD1 via yeast surface displaying peptide fragments
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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。 Objective：To analyze the immunogenicity of the extracellular region of Δ42PD1.Methods：Six fragments ofΔ42PD1 extracellular region-encoding sequence were amplified by PCR,and were cloned into p CTCON2 vector,a yeast surface displaying vector.Yeast cells were transfected with Δ42PD1 fragment-carrying plasmids,then yeast cells were spread on SDCAA plates.Single cell clones were selected and cultured in SGCAA media to induce expression of the target genes.Mouse anti-human Δ42PD1 anti-serum were generated by immunization of BALB/c mice via intramuscular injection of Δ42PD1-carrying plasmid plus in-situ electroporation.The binding of anti-serum with yeast cells surface-displaying Δ42PD1 fragments were analyzed using flowcytometry.Results：Nucleotide sequences analysis indicated that the amplified six fragments of Δ42PD1 sequence length were 110 bp,and the isolated sequence of Δ42PD1 fragments were 100% homology with PD1 gene previously registered in Gen Bank.Results from flowcytometry showed that among the six fragments of Δ42PD1 displaying on the surface of yeast cells,F3 and F2 profoundly bound Δ42PD1-specific polyclonal antibodies.Conclusion：F3 and F2 of Δ42PD1 is an immunogenic dominant region,which pave the way for generation ofΔ42PD1-specific monoclonal antibody and epitope mapping.
作者 程林 王子乔 徐六妹 唐娴 周泱 王辉
机构地区 深圳市第三人民医院
出处 《中国免疫学杂志》 CAS CSCD 北大核心 2016年第9期1333-1337,共5页 Chinese Journal of Immunology
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
关键词 程序性细胞死亡受体1 Delta-42程序性细胞死亡受体1 酵母表面展示 免疫原性 流式细胞术 PD1 Δ42PD1 Yeast surface display Immunogenicity Flowcytometry
分类号 R392.11 [医药卫生—免疫学]
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参考文献19

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中国免疫学杂志
2016年 第9期

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