Human Fl Pichia Pastoris Expression And Adenovirus-mediated Immune Gene Therapy

FL (flt3 ligand) is a recently identified cytokine that play important roles in the proliferation, survival, and differentiation of early murine and human hematopoietic stem cells and primitive uncommitted progenitor cells. Similar to SCF, FL has low biological activity alone, but it shows strong synergistic roles with other cytokines. A major difference between SCF and FL appears to be their effects on mast cells. SCF stimulates both the proliferation and activation of mast cells, while FL has no effect on mast cells. FL effects are not species-specific. Most importantly , FL can markedly augment the number of functional dendritic and NK cells in lymphoid and non-lymphoid tissues. Administration of FL results in a reversible accumulation of functionally active DCs in both lymphoid and nonlymphoid tissues. DCs are powerful antigen-presenting cells and show a remarkable capacity to stimulate antigen-specific T-cell responses. FL exerts anti-tumor activity in various experimental models. These functions have suggested a number of potential clinical benefits of FL in SCT and cancer immunotherapy. hFL was expressed in our lab by Pichia pastoris , and the possible antitumor effect of the rhFL on murine lymhphoma models were evaluated. The results provide further experimental evidences for the function of hFL in anti-tumor immunotherapy. This article mainly include the following two parts:First, the out-membrane domain of human FL K84E/Q122R mutant was expressed in Pichia pastoris. The codons for the 84^th and 122^th amino acids of hFL were changed to those for E and R, and N terminal codons were changed to the optimal ones for Pichia pastoris to elevate the expression level and to increase the biological activity. The recombinant plasmid pPIC9K-hFL was constructed and electrotransfonned into Pichia pastoris. Mut⁺ and Mut^s Pichia pastoris transformants expressing high-level hFL were obtained using 'in situ two-film method' and the best expression conditions were singled out: the Mut⁺ and Mut^s transformants expressed hFL best in BMMY (pH 6.0) and BMM (pH 6.0) respectively, with 2% methanol, induced for 96h. Compared with the Mut⁺, the Mut^s-hFL transformants showed higher expression level with less coloring contamination in the product and easier purification for hFL. The expression level reached 30mg/L. After purification by anion-exchange column chromatography and gel filtration chromatography, the purity of hFL is over 98%. About 20mg/L purified hFL could be obtained. After removal of N-linked carbohydrates by EndoH, the change of hFL MW indicates that hFL...