Cloning,expression and function of the extracellular fragment of human TRAIL gene  

作　　者：Yu GANG WANG KUN PENG ZHAO Ju GAO CHEN YAN LI YUANG FANG MA BEI FEN SHEN 

机构地区：[1]institute of Basic Medical Sciences, Beijing, P. R. China [2]Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng, P. R. China

出　　处：《Journal of Microbiology and Immunology》2007年第2期85-89,共5页中华微生物学和免疫学（英文版）

摘　　要：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.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 mononu- clear cells(PBMC)by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis.The expres- sion 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 MTT assay.PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-in- duced 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 prolif- eration 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.

关 键 词：TRAIL Prokaryotic expression Apoptosis 

分 类 号：Q78[生物学—分子生物学]