Cloning, Co-expression And Function Of Human Complement Regulatory Proteins

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 aglycophospholipid(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.