To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells （PBMC）...To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells （PBMC） by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis. The expression vector pET-30a/TRAIL was then constructed by DNA recombination method with a His-tag gene at the front of the TRAIL fragment, and the recombinant protein was expressed in E. coli BL21 （DE3）. Meanwhile, the expressed target protein was purified with Ni-NTA chromatography column and identified by SDS-PAGE and Western blotting. The proliferation inhibition activity of TRAIL-His was detected by MTF assay. PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-induced cell apoptosis. It was demonstrated that the target protein expressed in E. coli BL21 showed the same relative molecular mass as that the protein expected and could be recognized by both the anti-TRAIL polyclonal antibody and anti-His monoclonal antibody. In addition, this protein could also inhibit proliferation of human lymphoma cell line Jurkat cells or induce apoptosis of this cell line. It is apparent that a recombinant soluble TRAIL protein with biological activity is obtained and this prospective study can lay solid foundation for further research on the biological activity and application in the anti-tumor therapy.展开更多
文摘To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells （PBMC） by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis. The expression vector pET-30a/TRAIL was then constructed by DNA recombination method with a His-tag gene at the front of the TRAIL fragment, and the recombinant protein was expressed in E. coli BL21 （DE3）. Meanwhile, the expressed target protein was purified with Ni-NTA chromatography column and identified by SDS-PAGE and Western blotting. The proliferation inhibition activity of TRAIL-His was detected by MTF assay. PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-induced cell apoptosis. It was demonstrated that the target protein expressed in E. coli BL21 showed the same relative molecular mass as that the protein expected and could be recognized by both the anti-TRAIL polyclonal antibody and anti-His monoclonal antibody. In addition, this protein could also inhibit proliferation of human lymphoma cell line Jurkat cells or induce apoptosis of this cell line. It is apparent that a recombinant soluble TRAIL protein with biological activity is obtained and this prospective study can lay solid foundation for further research on the biological activity and application in the anti-tumor therapy.
