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| Construction and Expression of Prokaryotic Expression Vector of Soluble TNF-related Apoptosis Inducing Ligand and Its Anti-tumor Activity |
| WANG Shi-qi, LIU Jing-ying, LIU Cheng-lang, LI Chun, HU Xiao-feng, XIA Li-qiu, ZHANG You-ming |
| State Key Laboratory Breeding Base of Molecular Microbial Biology, College of Life Science, Hunan Normal University, Changsha 410081, China |
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Abstract Purpose:To study the induced expression condition and anti-tumor activity of protein sTRAIL from human HL-60 cell. Methods:Genomic RNA was extract from the human HL-60 cell. The sTrail gene segment was amplified with RT-PCR and cloned into the prokaryotic expression vector pET28a. The final expression vector pET28a-sTrail was transformed into E.coli BL21(DE3) for sTRAIL expression. Through IPTG induced, SDS-PAGE and LC-MS were used to determine the correct expression of sTRAIL. After purification by nickel-affinity chromatography, the recombinant sTRAIL was used to HUVEC, HeLa, Hep-3B, HCT-116, MDA-MB-231 and H460 cells. Result:The recombinant plasmid pET28a-sTrail was successfully built by the analysis of DNA sequencing. The result of SDS-PAGE show that sTRAIL gene was successfully expressed in E.coli BL21(DE3) and identified by LC-MS. The purified sTRAIL inhibited the growth of HeLa, HCT-116, MDA-MB-231, H460 and Hep-3B cell while not HUVEC. Flow cytometry showed the inducing apoptosis of Hep-3B cells by sTRAIL. Conclution:The prokaryotic expression vector of sTRAIL was successfully built, and the purified protein sTRAIL exhibited anti-tumor activity. This provided an important foundation for using sTRAIL protein as a antitumor drugs of clinical treatment.
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Received: 15 April 2015
Published: 22 December 2015
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