【摘要】 目的采用绿色荧光蛋白标记技术,研究细胞毒淋巴细胞相关抗原4(CTLA-4)靶向DNA疫苗编码抗原在细胞中表达的一般形式,并探讨CTLA-4融合抗原片段大小对靶向DNA疫苗在细胞中表达水平的影响。方法从靶向融合防龋DNA疫苗pGJA-P中去掉A-P片段,构建出靶向防龋质粒pGJGLU;以pVAX1质粒骨架替代pGJA-P和pGJGLU质粒中的pCI载体骨架,构建出pGJA-P/VAX和pGJGLU/VAX真核表达质粒;切下pGJGLU质粒的CTLA-4-Ig-GLU片段,克隆入pEGFP-N1中,获得pGJGLU/GFP质粒,转染CHO细胞,荧光显微镜下观察蛋白表达;将pGJA-P/VAX、pGJGLU/VAX和pGJGLU/GFP转染CHO细胞,以ELISA法检测培养上清液中融合蛋白的表达水平。结果pGJGLU/GFP转染细胞镜下表达特异性绿色荧光物质,呈颗粒状;pGJA-P/VAX、pGJGLU/VAX和pGJGLU/GFP3种质粒可以表达融合蛋白并分泌到培养上清液中,且所表达的融合蛋白浓度不同,并以pGJGLU/VAX表达的水平最高。结论CTLA-4融合蛋白可以以分泌形式表达,CTLA-4靶向疫苗编码的融合抗原片段大小影响其在细胞中的表达分泌水平。更多还原

【Abstract】 Objective To investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells. Methods The A-P fragment of PAc (surface protein antigen)was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-Ⅰ of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants. Results Specific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX、pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX. Conclusions CTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.更多还原