Studies On Plasmid Stability Of Recombinant Anabaena Sp. PCC7120 And Optimization Of Fermentation Conditions Of RhTNF-α Expression

Human tumor necrosis factor alpha(hTNF- α ) is a cytokine produced by macrophages. With varied bio-activities and functions like anti-tumor, anti-virus and immunoregulation, hTNF- α has wide and significant clinical application prospects for cancer treatment Because of limited sources from nature, the studies on recombinant TNF and its derivatives have been developing very quickly. Cyanobacteria have many advantages, which make them a very potentially ideal host cell system, e.g. containing abundantly various health ingredients, free from toxic proteins, easy cultivation and commercialization.Former researchers have transferred the hTNF- α gene into Anabaena sp. PCC7120. In order to get better economic benefits, the dissertation focused on recombinant plasmid stability as well as effect of photoheterotrophic culture and environmental factors on growth and TNF expression, with the aim of improving TNF expression yield and advancing its industrialization process. The results are as fellows:Short-term preservation method for Anabaena sp. PCC7120 was investigated, cryoprotective agents could enhance low temperature(4℃) resistance capability of Anabaena sp. PCC7120 cells. Among these agents, 4.0% dimethyl sulfoxide with 4.0% glycerol had the best effect It's good for the next preservation step by dripping homogeneously the Anabaena sp. PCC7120 solution in slats during inoculation. Adding little aseptic water regularly to slats could prolong preservation time.The growth rate of Anabaena sp. PCC7120 would slow down upon transfer of foreign plasmid. At the growth curve, the time of reaching the maximum TNF activity (6.735 ×10~4U per mg cells) was at around the sixteenth day, which was different from the optimum biomass harvest time. TNF activity decreased gradually afterwards. Adding neomycin would bring certain effect on growth of recombinant Anabaena sp. PCC7120. A sensitive kinetic equation for neomycin in wild type Anabaena sp. PCC7120 was set up, even if 1μg/m(?) was enough to kill the cells of wild type Anabaena sp. PCC7120.The segregative and structural stability of recombinant plasmid PDC-TNF was...