Construction of prokaryotic expression vector of human soluble TNF-related apoptosis inducing ligand and studies of the expression, purification and activity identification of recombinant protein
Objective To construct the TNF-related apoptosis inducing ligand ( TRAIL) gene prokaryotic expression vector expressing soluble protein with high activity. Methods Genomic RNA was extracted from the peripheral blood of health adults. The extracellular domain region of TRAIL gene was amplified with RT-PCR and cloned into the prokaryotic expression vector pET30a. The recombinant was confirmed by EcoR I/ Xho I digestion, PCR, and DNA sequencing. Meanwhile, the recombinant was induced into E. coli BL21. Recombinant protein was analyzed by SDS-PAGE and Western blot. Huh-7 cells were stimulated by recombinant protein and standard protein respectively to compare their bioactivity by CCK8 and FCM. Result pET30a/TRAIL was successfully constructed and soluable His-rTRAIL could be expressed and identified by SDS-PAGE and Western blot analysis. The results of CCK8 and FCM showed that His-rTRAIL could induce apoptosis of Hch-7 cells. Conclusion A prokaryotic expression vector pET30a/TRAIL was constructed successfully, which expressed soluble protein with high bioactivity. This provided a new idea for tumor therapy.
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