草鱼CD8α、CD207基因重组真核表达质粒的构建与表达分析

Construction and expression analysis of recombinant eukaryotic expression plasmids of CD8α and CD207 genes in Ctenopharyngodon idella

摘要:

为进一步研究CD8α、CD207在草鱼树突状细胞 (dendritic cells，DCs)中的功能，实验构建了草鱼CD8α、CD207基因重组真核表达质粒并在草鱼DCs中成功表达。采用逆转录-聚合酶链式反应 (RT-PCR)技术从草鱼DCs中获得CD8α、CD207开放阅读框序列 (ORF)，分别插入真核表达质粒pcDNA3.1，构建重组真核表达质粒pcDNA3.1-CD8α、pcDNA3.1-CD207；经测序鉴定正确后，采用脂质体法将重组真核表达质粒分别转染至草鱼树突状细胞，经细胞免疫荧光技术和蛋白免疫印迹 (Western blot) 法验证CD8α、CD207的表达。结果显示，在蛋白免疫印迹实验中CD8α、CD207蛋白可正常表达，且大小与预测结果一致。细胞免疫荧光结果显示，CD8α、CD207蛋白主要在草鱼DCs的细胞膜上表达。本研究成功构建pcDNA3.1-CD8α、pcDNA3.1-CD207重组真核表达质粒，为后续研究CD8α、CD207基因在DCs中的作用机制奠定了基础。

关键词:草鱼;树突状细胞;CD8α;CD207;真核表达质粒

Abstract:

Dendritic cells are the outposts of the immune system, which link innate immunity with adaptive immunity, and thus are considered as the most powerful antigen-presenting cells in vivo. To further investigate the functions of CD8α and CD207 in grass carp (Ctenopharyngodon idella) dendritic cells (DCs), the recombinant eukaryotic expression plasmids of CD8α and CD207 genes were constructed and successfully expressed in DCs of C. idella. The open reading frames (ORFs) of CD8α and CD207 were obtained from DCs of C. idella by RT-PCR and inserted into recombinant eukaryotic expression plasmid pcDNA3.1, respectively, to construct recombinant eukaryotic expression plasmids pcDNA3.1-CD8α and pcDNA3.1-CD207; after the sequencing was confirmed to be correct, the recombinant eukaryotic expression plasmids were transfected into DCs of C. idella using liposome method, and the expressions of CD8α and CD207 were verified by cell immunofluorescence technology and Western blot. The results showed that in the Western blot assay, the CD8α and CD207 proteins were expressed normally and significantly overexpressed after transfection of the recombinant eukaryotic expression plasmid, and the size was consistent with that of the predicted result. Cellular immunofluorescence showed that the CD8α and CD207 proteins were mainly expressed on the cell membrane of DCs. Hence, DCs of C. idella exhibit immunophenotypes and functions that are conserved in their mammalian counterparts. The recombinant eukaryotic expression plasmids of pcDNA3.1-CD8α and pcDNA3.1-CD207 were successfully constructed, which laid a foundation for further research on the action mechanism of CD8α and CD207 genes in DCs. Carrying out studies on the immunophenotype of DCs will play a positive role in analyzing the DCs-mediated immune regulation mechanism of C. idella. In the cultivation process, C. idella are easily infected by pathogens and induce inflammatory reactions. The development of DCs-based immunotherapy can provide new ideas for the prevention and control of C. idella related diseases, which has a wide application prospect in reducing diseases and enhancing the immune efficacy of vaccines for teleost fish.

Key words:Ctenopharyngodon idella;dendritic cells;CD8α;CD207;eukaryotic expression plasmid