人膜补体调节蛋白MCP、CD59在稳定转染的细胞中的共表达及功能研究

采取内核糖体进入位点(IRES)策略构建含人的膜补体调节蛋白基因MCP和CD59的cDNA的双顺反子真核表达载体pcDNA3-MCPIRESCD59,以磷酸钙沉淀法转染NIH3T3细胞,用 G418筛选阳性克隆,并研究MCP和CD59双基因在稳定细胞系中的共表达及保护功能.PCR实验结果显示双基因稳定整合在异源细胞NIH3T3的染色体上,RT-PCR及Western印迹实验分别从 RNA水平和蛋白质水平证实了人补体调节蛋白分子MCP和CD59在细胞系中皆获得同步表达.检测连续传代30次的NIH3T3 pcDNA3-MCPIRESCD59,结果表明人MCP和CD59基因仍稳定整合在细胞基因组中,并未随着传代而丢失,为稳定的转双基因细胞系.补体依赖的细胞毒反应表明,pcDNA3-MCPIRESCD59转染细胞由于MCP和CD59的共表达获得了高于MCP或CD59单一表达时所提供的保护功效,能更好地抑制人补体依赖的细胞毒作用的发生,保护宿主细胞免受人补体的攻击.以上结果表明,所构建的双基因重组表达载体实现了不同人补体调节蛋白基因高效转移和高水平共表达,在克服超急性排斥反应的基因治疗中有潜在的应用价值.

Studies on co-expression and function of human complement regulatory proteins MCP and CD59 in stably transfected NIH3T3 cells

Dicistronic expression vector pcDNA3-MCPIRESCD59 was constructed successfully by the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV). After transfected into NIH3T3 cells with the calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-MCPIRESCD59 transfectant was obtained by G418 selection. Extraneous genes integration was identified by PCR. RT-PCR and Western blot analysis demonstrate that the EMCV IRES allows for efficient co-expression of human CD59 and MCP in NIH3T3 cells transfectant. Human MCP and CD59 cDNA are still integrated in NIH3T3 genomic DNA after continuous 30 times' passages, indicating that NIH3T3 pcDNA3-MCPIRESCD59 transfectant is a stable cell line. Human complement-mediated cytolysis assays show that NIH3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-MCP, and pcDNA3-MCPIRESCD59 are protected from C-mediated damage, and co-expressed human MCP and CD59 provide more excellent protection against C-mediated attack as compared with either CD59 or MCP expressed alone. These results suggest that the two dicistronic vector represents an effective and efficacy strategy to overcome C-mediated damage and has the potential therapeutic value for effectively controlling complement activation and finally for preventing hyperacute rejection in clinical gene therapy.