人补体调节蛋白基因的克隆、共表达及功能研究
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摘要
异种器官移植成为解决器官短缺的有效途径正受到越来越多的关注。然而免疫排斥反应,特别是超急性排斥反应(hyperacute rejection,HAR)很大程度上制约了它的发展。补体系统激活是异种器官移植超急性排斥反应发生的主要原因,抑制受体的补体系统就可以阻止超急性排斥反应的发生。膜补体调节蛋白衰变加速因子(decay accelerating factor,DAF),膜辅蛋白(membrane cofactor protein,MCP)和保护素(protectin,CD59)是补体系统中具有明显的同种限制作用的3种补体下调因子,在补体攻击时能提供区分“自我”与“非我”的方式,保护宿主细胞不受同源性补体的伤害,从而延长移植物的存活时间,克服超急性排斥反应的发生,在异种器官移植方面有重大的应用价值。因此人补体调节蛋白(complement regulator proteins,CRPs)是目前异种移植界的研究热点之一。
本研究根据文献报道的衰变加速因子DAF cDNA的序列,设计并合成特异引物,以中国人胚胎mRNA为模板,采用RT-PCR方法扩增出1684 bpDAF cDNA。序列分析表明,该cDNA含DAF全长编码区,共编码381个氨基酸,没有编码Alu家族的序列,为GPI锚连型DAF。
将DAF全长cDNA序列插入真核表达载体pcDNA3,成功地构建了重组真核表达载体pcDNA3-DAF,以磷酸钙沉淀法转染NIH/3T3细胞,用G418筛选获得NIH/3T3DAF,PCR实验结果显示DAF基因整合在转化的NIH/3T3细胞的染色体上,RT-PCR和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DAF在NIH/3T3DAF细胞系中获得表达。检测连续传代30次NIH/3T3DAF结果表明人补体调节蛋白基因DAF仍稳定整合并高效表达,并未随着传代而丢失,以上结果显
Xenotransplantation, as an alternative approach for patients with end stage organ failure, has attracted more and more attention because of the serious shortage of human organs for allotransplantaion. However, progress towards its clinical application has been hampered by the phenomenon of hyperacute rejection (HAR), which occurs almost immediately upon reperfusion of the transplanted organ and leads dramatic and irreversible graft destruction within minutes to hours. The activation of complement is one of critical events in the pathogenesis of HAR. The complement regulatory proteins (CRPs) such as membrane protein DAF, MCP and CD59 had function in a species-restricted manner, which could regulate and control the activation of the complement system strictly. They are important for protecting host cells from damage by complement. It has been demonstrated that xenografts expressing these proteins could escape complement damage and prolong their survival time. Moreover, combined applications of different hCRPs represented potential methods for achieving total complement inhibition, and thereby increasing the possibility of inter-species transplantation for clinical experiments. Co-expression of hCRPs would be an effective strategy to help overcome host C-induced hyperacute rejection.In this work, primers specific for decay accelerating factor DAF cDNA were designed and synthesized according to the previous report. 1684bp human DAF cDNA containing full encoding region were cloned by reverse transcription PCR from the total mRNA of the Chinese human embryo. Sequence analysis showed that DAF cDNA included 381aa open reading frame and was lack of Alu family sequence, which indicated that it was a
glycophospholipid(GPI)-anchored molecule.Based on the above work, human DAF cDNA was cloned into pcDNA3 vector and transfected into NIH/3T3 cells to generate stable cell lines by G418 screening. Extraneous genes integration and expression were identified by PCR, RT-PCR, Western blot and immunofluorescence microscopic analysis. The transfected cells were tested for its susceptibility to human C-mediated cytolysis, indicating that the DAF gene could be expressed in xenogenetic cells stably to confer protection against human serum damage.Furthermore, we constructed serial dicistronic mammalian expression vectors by using independent promoters or the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV) as follows:pcDNA3-MCPCD59-DP, pcDNA3-DAFCD59-DP, pcDNA3-DAFMCP-DP, pcDNA3-MCPIRESCD59, pcDNA3-MCP-IRES DAF and pcDNA3-CD59IRESDAF. After transfected into NIH/3T3 cells with the calcium phosphate precipitation method, the stable expression clones were obtained by G418 screening. Extraneous genes integration and co-expression were identified by PCR, RT-PCR, Western blot and immunofluorescence microscopic analysis. All these results demonstrated that the two strategies of the internal promoter and the EMCV IRES allowed for efficient co-expression of different human complement regulatory proteins on the surface of transfected NIH/3T3 cells. Human complement-mediated cytolysis assays showed that double hCRPs exhibited more protection against cytolysis by human serum compared to the cells with only one expressed alone. Moreover, co-expressed DAF-CD59 or DAF-MCP proteins could provide fully protection against C-mediated damage by their synchronous action, indicating that these combinations could be used to produce transgenic pigs for xenotransplantation.All above results suggested that these two kinds of polycistronic vectors should improve the efficiency and effectiveness of multi-gene delivery and these vectors have potential therapeutic value for effectively controlling complement activation and finally for preventing hyperacute rejection in clinical gene therapy.
本研究根据文献报道的衰变加速因子DAF cDNA的序列,设计并合成特异引物,以中国人胚胎mRNA为模板,采用RT-PCR方法扩增出1684 bpDAF cDNA。序列分析表明,该cDNA含DAF全长编码区,共编码381个氨基酸,没有编码Alu家族的序列,为GPI锚连型DAF。
将DAF全长cDNA序列插入真核表达载体pcDNA3,成功地构建了重组真核表达载体pcDNA3-DAF,以磷酸钙沉淀法转染NIH/3T3细胞,用G418筛选获得NIH/3T3DAF,PCR实验结果显示DAF基因整合在转化的NIH/3T3细胞的染色体上,RT-PCR和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DAF在NIH/3T3DAF细胞系中获得表达。检测连续传代30次NIH/3T3DAF结果表明人补体调节蛋白基因DAF仍稳定整合并高效表达,并未随着传代而丢失,以上结果显
Xenotransplantation, as an alternative approach for patients with end stage organ failure, has attracted more and more attention because of the serious shortage of human organs for allotransplantaion. However, progress towards its clinical application has been hampered by the phenomenon of hyperacute rejection (HAR), which occurs almost immediately upon reperfusion of the transplanted organ and leads dramatic and irreversible graft destruction within minutes to hours. The activation of complement is one of critical events in the pathogenesis of HAR. The complement regulatory proteins (CRPs) such as membrane protein DAF, MCP and CD59 had function in a species-restricted manner, which could regulate and control the activation of the complement system strictly. They are important for protecting host cells from damage by complement. It has been demonstrated that xenografts expressing these proteins could escape complement damage and prolong their survival time. Moreover, combined applications of different hCRPs represented potential methods for achieving total complement inhibition, and thereby increasing the possibility of inter-species transplantation for clinical experiments. Co-expression of hCRPs would be an effective strategy to help overcome host C-induced hyperacute rejection.In this work, primers specific for decay accelerating factor DAF cDNA were designed and synthesized according to the previous report. 1684bp human DAF cDNA containing full encoding region were cloned by reverse transcription PCR from the total mRNA of the Chinese human embryo. Sequence analysis showed that DAF cDNA included 381aa open reading frame and was lack of Alu family sequence, which indicated that it was a
glycophospholipid(GPI)-anchored molecule.Based on the above work, human DAF cDNA was cloned into pcDNA3 vector and transfected into NIH/3T3 cells to generate stable cell lines by G418 screening. Extraneous genes integration and expression were identified by PCR, RT-PCR, Western blot and immunofluorescence microscopic analysis. The transfected cells were tested for its susceptibility to human C-mediated cytolysis, indicating that the DAF gene could be expressed in xenogenetic cells stably to confer protection against human serum damage.Furthermore, we constructed serial dicistronic mammalian expression vectors by using independent promoters or the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV) as follows:pcDNA3-MCPCD59-DP, pcDNA3-DAFCD59-DP, pcDNA3-DAFMCP-DP, pcDNA3-MCPIRESCD59, pcDNA3-MCP-IRES DAF and pcDNA3-CD59IRESDAF. After transfected into NIH/3T3 cells with the calcium phosphate precipitation method, the stable expression clones were obtained by G418 screening. Extraneous genes integration and co-expression were identified by PCR, RT-PCR, Western blot and immunofluorescence microscopic analysis. All these results demonstrated that the two strategies of the internal promoter and the EMCV IRES allowed for efficient co-expression of different human complement regulatory proteins on the surface of transfected NIH/3T3 cells. Human complement-mediated cytolysis assays showed that double hCRPs exhibited more protection against cytolysis by human serum compared to the cells with only one expressed alone. Moreover, co-expressed DAF-CD59 or DAF-MCP proteins could provide fully protection against C-mediated damage by their synchronous action, indicating that these combinations could be used to produce transgenic pigs for xenotransplantation.All above results suggested that these two kinds of polycistronic vectors should improve the efficiency and effectiveness of multi-gene delivery and these vectors have potential therapeutic value for effectively controlling complement activation and finally for preventing hyperacute rejection in clinical gene therapy.
引文
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