人补体调节蛋白基因的克

发布时间：2018-04-15 10:06

本文选题：超急性排斥反应 + 补体调节蛋白　；参考：《武汉大学》2005年博士论文

【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
[Abstract]:More and more attention has been paid to xenotransplantation as an effective way to solve organ shortage.However, immune rejection, especially hyperacute rejection (HARs), restricts its development to a large extent.The activation of complement system is the main cause of hyperacute rejection in xenotransplantation. The suppressive complement system can prevent the occurrence of hyperacute rejection.The decay accelerating factor-dafs, membrane coprotein membrane cofactor protein (MCPs) and protectinin CD59) are three kinds of complement down-regulation factors which have the same limiting effect in the complement system.During complement attack, it can provide a way to distinguish "self" from "non-self" and protect host cells from the damage of homologous complement, thus prolonging the survival time of grafts and overcoming the occurrence of hyperacute rejection.It has great application value in xenogeneic organ transplantation.Therefore, human complement regulator proteins (CRPs) is one of the hotspots in the field of xenotransplantation.Based on the sequence of decay acceleration factor (DAF cDNA) reported in the literature, a specific primer was designed and synthesized. 1684 bpDAF cDNA was amplified by RT-PCR using Chinese embryonic mRNA as a template.Sequence analysis showed that the cDNA contained a full-length coding region of DAF, encoding 381 amino acids, but not encoding the sequence of Alu family. It was a GPI anchor type.The full-length cDNA sequence of DAF was inserted into eukaryotic expression vector pcDNA3. The recombinant eukaryotic expression vector pcDNA3-DAF was successfully constructed and transfected into NIH/3T3 cells by calcium phosphate precipitation.The results of NIH / 3T3DAFN PCR showed that the expression of human complement regulatory protein DAF in NIH/3T3DAF cell line was confirmed by RT-PCR and Western blotting from the RNA level and protein level, respectively.The results of 30 successive passages of NIH/3T3DAF showed that the human complement regulatory protein gene DAF was still stable and highly expressed, and was not lost with passage.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q78;R392

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