人补体调节蛋白MCP、CD59共表达体系的构建及其功能研究  

Studies on Establishment of Co-expression System and Function of Human Complement Regulatory Proteins MCP and CD59

作　　者：徐莉[1] 赵舟宙[1] 刘辉[1] 李文鑫[1] 

机构地区：[1]武汉大学生命科学学院病毒学国家重点实验室,武汉430072

出　　处：《中国生物工程杂志》2007年第9期8-13,共6页China Biotechnology

基　　金：国家"863"计划资助项目(2001AA216071)

摘　　要：利用双启动子构建含人补体调节蛋白MCP和CD59 cDNA的双顺反子重组表达载体pcDNA3-MCPCD59-DP,以磷酸钙沉淀法转染NIH3T3细胞,用G418筛选获得NIH3T3pcDNA3-MCPCD59-DP转化细胞。PCR实验结果显示人MCP和CD59整合在转化的NIH3T3细胞的染色体上,RT-PCR和Western blot实验分别从RNA水平和蛋白质水平证实了人MCP和CD59在转化细胞中的共表达。检测连续传代30次的NIH3T3pcDNA3-MCPCD59-DP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。补体溶破实验表明,pcDNA3-MCPCD59-DP转染细胞由于人MCP和CD59的共表达获得了较MCP或CD59单一表达时更好的保护功效,能有效地保护NIH3T3细胞免受人补体的攻击,从而抑制补体依赖的细胞毒反应的发生。以上结果表明所构建的双基因重组表达载体实现了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。Recombinant expression vector pcDNA3-MCPCD59-DP containing human membrane complement regulatory proteins （hCRPs） MCP and CD59 eDNA was constructed successfully by using two independent promoters. After transfected into NIH3T3 ceils with calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-MCPCD59-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human CD59 and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysisl Human MCP and CD59 eDNA were integrated in NIH3T3 pcDNA3- MCPCD59-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-MCPCD59-DP were stable cell lines. Human complement-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-MCP, and pcDNA3-MCPCD59-DP were protected from C-mediated damage and co-expressed human MCP and CD59 provided more excellent protection against C-mediated attack as compared with either CD59 or MCP expressed alone. The dicistronic vector represents an effective and efficacy strategy to overcome C-mediated damage and has potential＇ therapeutic value for effectively controlling complement activation and finally for preventing hyperacute rejection in clinical gene therapy.

关 键 词：人补体调节蛋白 双基因重组表达载体 共表达 超急性排斥反应 

分 类 号：Q789[生物学—分子生物学]