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

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

YU GANG WANG; KUN PENG ZHAO; JU GAO CHEN; YAN LI; YUANG FANG MA; BEI FEN SHEN

【期刊名称】《《中华微生物学和免疫学杂志（英文版）》》 【年(卷),期】2007(005)002

【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 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