TNFR1及其突变体重组质粒的构建及肿瘤生物学功能的研究

目的]构建TNFR1及其突变体重组质粒,研究TNFR1胞浆段结构域与sTNF-α肿瘤生物学功能的关系.方法]通过RT-PCR和重叠PCR,构建TNFR 1-pEGFP-N1,Y236A-TNFR1-pEGFP-N1、ΔNSD-TNFR1-pEGFP-N1和△DD-TN FR 1-pEGFP-N1.采用荧光显微镜观察...

Construction of Recombined Plasmids of TNFR1 and Its Mutants and Their Tumor Bio-functions

Purpose] To construct TNFR1 recombinant plasmids of TNFR1 and its related mutants,and to study the relationship between the TNFR1 cytoplasm domains and the anti-tumor biological function of sTNF-α.Methods] The TNFR1-pEGFP-N1,Y236A-TNFR1-pEGFP-N1,ΔNSD-TNFR1-pEGFP-N1 and ΔDD-TNFR-pEGFP-N1 recombinant plasmids were constructed by RT-PCR and overlapping PCR.TNFR1 internalization was observed with fluorescence microscope.The TNFR1 expression rate was detected by flow cytometry and cytotoxicity was detected by MTT colorimetric.Results] Recombinant plasmids of TNFR1 full length gene and the related mutants were successfully obtained without point mutation,frame-shift or deletion mutation.With these vectors as tools,the results indicated that TNFR1 wildtype can mediate sTNF-α internalization whereas Y236A-TNFR1 can＇t.It was shown that ΔNSD-TNFR1 and ΔDD-TNFR1 also affected the cytotoxicity of sTNF-α to target cell.Conclusion] TNFR1 internalization domain,NSD and DD domain are essential for the cytotoxicity of sTNF-α against tumor cells.This study will help us to further understand the relationship between the TNFR1 and sTNF-α mediated tumor cytotoxicity,it will also provide the experimental tools to elucidate the mechanism of tmTNF-α cytotoxicity against tumor cells,so as to provide the novel target for tumor gene therapy.