Optimization And Control Of Growth And Exogenous Gene Expression Of Recombinant Anabaena Sp. PCC7120

Human tumor necrosis factor alpha ( hTNF — α ) has a great variety of bio-activities. With the development of research, it is possible to produce low toxic and high effective hTNF- α and its derivatives which has wide clinical application prospects in cancer treatment. Former researchers have transferred the hTNF- α gene into Anabaena sp. PCC7120 and the gene have been expressed. In order to increase cell density and exogenous gene expression level, this dissertation mainly studied on the stability of recombinant plasmid, the optimization and control of the growth and exogenous gene expression of recombinant Anabaena sp. PCC7120. The results as fellows:The relationship between biomass of recombinant Anabaena sp. PCC7120 and culture solution absorbency was investigated at first. The wavelength and measure scale of spectrophotometry, standard curve of the biomass was determined.The growth rate of recombinant Anabaena sp. PCC7120 approach to the growth rate of wild Anabaena sp. PCC7120 closely. The experiment proved that recombinant Anabaena sp. PCC7120 could remain segregative stability of recombinant plasmid pDC-TNF. Through comparing the growth and exogenous gene expression of recombinant Anabaena sp. PCC7120 that existed from generation to generation without neomycin and cultured in different medium, it is also proved that the structural stability of recombinant plasmid pDC-TNF was high, but the copy number of recombinant plasmid pDC-TNF would reduce if recombinant Anabaena sp. PCC7120 was cultured in medium without neomycin. The neomycin in the seed medium could hold the stability of pDC-TNF during culture process, so the quantity of neomycin used in fermentation culture could be reduced much.The growth and exogenous gene expression of recombinant Anabaena sp. PCC7120 could be significantly improved under mixotrophic culture conditions. When sucrose and NaN03 were 9g/L and 2.25g/L respectively, inoculum volume was 5%, light intensity was 1000Lux, light/dark cycle was 12hr/12hr, temperature was 25-30℃, and 30mL medium was added in 100mL shake flask, it was optimum for the...