| Human interleukin-15 (IL-15) is a regulative cytokine discovered by Grabstein et al in 1994. IL-15 is similar to IL-2 in multiple biological activities. IL-15 plays important roles in immune response, it induces the T, B and NK cell proliferation and differentiation, increase the CTL/NK cytotoxicity, and may have more potent anti-tumor activities than IL-2 in tumor immunotherapy.We obtained pure recombinant human interleukin-15 (rhIL-15) by gene engineering, protein expression and purification technology. The purity of final product was over than 95% by protein laser scanning analysis. The antitumor effect of rhIL-15 was studied on several kinds of mouse tumor models established on the basis of our previous work. Our work provided the basis for rhIL-15 to be developed into a promising new anti-tumor drug. This paper included three parts:1. Prokaryotic expression, purification and identification of rhIL-15.2. Preparation of mouse monoclonal antibody against rhIL-15.3. Studies on acute toxicity and anti-tumor effect of rhIL-15.1. Prokaryotic expression, purification and identification of rhIL-15A DNA fragment encoding human IL-15 mature protein was synthesized by using E. coli prefered genetic codons with overlapping PCR method. The sequence of hIL-15 gene synthesized is confirmed by DNA sequencing. The hIL-15 gene was inserted into prokaryotic expression vectors, such as pGEX-2T, pET30a, PET42, pETBlue-1 and PBV220, for non-fusion expression, respectively, and expressed in several E. coli strains. The high performance engineering bacterium strain, BL21 StarTM(DE3) plysS/ pBV220-hIL-15, was obtained. RhIL-15 was expressed as inclusion body in the engineering bacterium, and its highest yield was 15.2% of the total bacterium proteins. RhIL-15 was analyzed by western blot with mouse monoclonal antibody against human IL-15, and found that it could be recognized specifically by this monoclonal antibody. The rhIL-15 as inclusion body was... |
