Construction And Expression Of Prokaryotic Expression Vector Of Soluble TNF- Related Apoptosis Inducing Ligand And Its Anti-tumor Activity

TRAIL is a newly found member of TNF superfamily, and it is a protein molecule human body normal expressed. TRAIL specificly combine with natural receptors on the surface of the tumor cells, thus induce the apoptosis of tumor cell but not normal cells and tissues,compared to the founded apoptosis factor TNF-α which have serious inflammation. The key features of TRAIL protein make it become the hotspot in the study of tumor treatment. TRAIL protein contain 281 amino acid residues, and the low solubility of the complete protein molecules limits its application. The solubility of 114-281 amino acid of TRAIL protein is greatly increased, at the same time maintain the activity.Using microorganisms as the host has been attached great importance to express foreign protein of which represented by E.coli. The reason is that E. coli expression system has a clear background, large expression,simple purification and control. The research is becoming more and more attention.Enomic RNA was extracted from the human HL-60 cell. The sTrail gene segment was amplified with RT-PCR and cloned into the prokaryotic expression vector pET28 a. The final expression vector pET28a-sTrail was transformed into E.coli BL21(DE3) for sTRAIL expression after identification and sequencing correctly. Then the s TRAIL were expressed efficiently through IPTG induction. In the optimization of expressing condition of soluble protein, we identified when IPTG concentration is 0.1 mM, its induction time of 12 hours and the temperature is 16 ℃, the soluble protein’s production is highest.Through optimizing the nickel-affinity chromatography,SDS-PAGE and LC-MS, we determine the purified protein is sTRAIL. The recombinants TRAIL inhibited the growth of HeLa, HCT-116, MDA-MB-231, Hep-3B and H460 cell while not HUVEC. The results of MTT show that the inhibition ratio is concentration dependent, which is 27.2%, 29.7%,32.9%, 22.5% and 10.8% respectively for those cells above after theated in 1200 ng/mL s TRAIL for 48 hours. From the inverted phase contrast microscope observation found that a growing number of tumor cells change from the spindle or irregular form into round, thick and light transmittance change. At the same time, we also found that sTRAIL protein in normal human umbilical vein endothelial cells(HUVEC) has no cytotoxicity.We study the cloning of sTrail gene, sTRAIL expression,purification and anti-tumor activity. The sTRAIL protein has good antitumor biological activity, and harmless to normal cells, and it is expected to become new clinical antitumor drugs molecule. This study also provides a new experimental basis for the future work.