Vector Construction And Eukaryotic Expression Of Human GITR_aa1-165

Objective To construct containing human GITR_aal-165eukaryotic expression vector pDisplay-hGITR_aal-165,and an eukaryotic expression vector of hGITR_aal-165-IgG1 Fc /pcDNA3.1(+),then expressed the fusion protein in COS-7 cells.Methods(1)Extracellular region gene of human GITR was obtained by PCR method.Target gene was digested by Bglâ…¡and Pstâ… ,recovered from 1%agarose and ligated into eukaryotic expression vector pDisplay.The recombinant vector was transformed into E.coli DH5a.Positive clone would be detected by two restriction enzyme and sequencing analysis. Eukaryotic expression vector pDisplay- hGITR_aal-165was extracted.The COS-7 cells were transfected in the mediation of liposome mixed with pDisplay-hGITR_aal-165vector,and culture supernatant was collected at different time points.The recombinant protein was analyzed by Western blot.(2)The hGITR_aal-165gene was obtained by PCR amplification and inserted into expression vector hIgG1Fc-pcDNA3.1(+),which contained human IgG1-Fc gene.The hGITR_aal-165-IgG1 Fc/pcDNA3.1(+)vector was transformed into E.coli DH5a and identified by restriction digestion and sequencing analysis.Control plasmid eGFP-IgG1 Fc /pcDNA3.1(+)was constructed by the same method.Eukaryotic expression vector hGITR_aal-165-IgG1 Fc/pcDNA3.1(+)and control vector eGFP-IgG1 Fc /pcDNA3.1(+)were extracted.The COS-7 cells were transfected with liposome method,then culture supernatant was collected at different times. The fusion protein was analyzed by Western blot.THP-1,HUEVC cells were cultured with the fusion protein,and cells population were calculated at differene time points.Results(1)hGITR_aal-165gene was obtained by PCR.The eukaryotic expression vector of pDisplay-hGITR_aal-165was successfully constructed. The vectoe was identified by restriction digestion and PCR,and the sequencing analysis showed that hGITR_aal-165gene was correct.Expression of pDisplay-hGITR_aal-165protein in COS-7 cells was detected by Western blot and the MW of soluble protein was about 15KD.(2)The vector hGITR_aal-165-IgG1 Fc/pcDNA3.1(+)was successfully constructed and the inserted target gene shared 100%homology with the sequence of mRNA for human GITR in Genbank.Expression of control plasmid eGFP-IgG1 Fc/pcDNA3.1(+)in COS-7 cells was detected by fluorescence microscope, and the best ratio was 1:3,and the best collection time was 48h.Expression of hGITR_aal-165-IgG1 Fc/pcDNA3.1(+)fusion protein in COS-7 cells was detected by Western blot.Initial experiment result showed that THP-1 cell could be encouraged proliferation by the hGITR_aal-165-IgG1 Fc/pcDNA3.1(+) fusion protein.Conclusion The constructed recombinant vector pDisplay-hGITR_aal-165 could be used for the expression of hGITRaa_l-165protein in eukaryotic cells. The eukaryotic expression vector of hGITR_aal-165-IgG1 Fc/p cDNA3.1(+) was successfully constructed and expressed in COS-7 cells,providing research foundation for biology of GITR.