Optimization Of Expression System Transformed Human Lactoferrin In Desert Chlorella

Object: We transformed huamn lactoferrin gene into Desert Chlorella GTD8A1, In order to establish Desert Chlorella expression system of human lactoferrin and obtian transgenic Desert Chlorella GTD8A1-HLF; It was increased positive clones and cell survivl of transgenic Desert Chlorella GTD8A1-HLF that the electroporation conditions（breakdown voltage, cell cycle, osmosis buffer, osmosis time, plasmid, electroporation buffer） was optimizated; It was imprved transgenic Desert Chlorella GTD8A-HLF express content of human lactoferrin that the product conditions（p H, NaCO₃, NaNO₃, K₂HPO₄, Na Cl, MgSO₄, FeSO₄, ZnSO₄, CuSO₄, H₃BO₃） was optimizated; This method will provide some engineered micro-organism and conditional production to further use it to yield human lactoferrin in industrialization and commercialization.Methods:（1） Established the recombinant plasmid p CAMBIA2300-35S-GFP-HL F-OCS and human lactoferrin gene regard as reference gene of optimizatio n of electro –transformation conditions. The various parameters of cell viab ility and transfection efficiency were determined by single factor experimen ts. And then, the optimal electroporation conditions were obtained through orthogonal test.（2） To establish the recombinant plasmid p CAMBIA2300-35S-HLF-OCS, We transformed the plasmid p CAMBIA2300-35S-HLF-OCS into Desert Chlorella GTD8A1 by electroporation, Sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and Western Blot showed that human lactoferrin successfully epress in Desert Chlorella GTD8A1-HLF, this result showed that we have obtained transgenic Desert Chlorella GTD8A1-HLF.（3） The top and bottom limitation of Plackett- Burman（PB） was confirmed by single factor experiments, and then was going to Central Composite Design（CCD） experiments, The optimum of product conditions,with the RT-PCR and ELISA methods is detecte human lactoferrin concentration in Deserted chollera GTD8A1-HLF cells under the different culture conditions, was confired.Results and Conclusion:1. Results of the optimal electroporation conditions was showed: breakdown voltage 1400 V, cell cycle 8day, osmosis buffer 0.2mol/L, osmosis time 0h, plasmid 6μg/m L, and electroporation buffer 0.4mol/L. Under the optimal composition of electroporation conditions, the detected results of cell viability is 51.30%, and fluorescence positive rate is 52.54%, and PCR positive rate is 42.30%.2. The results was showed :The huaman Lactoferrin gene was transformed into the Deserted chollera GTD8A1 cells by the electroporation. The PCR, Sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and Western Blot showed that the recombinant human lactoferrin had a band of 70 k Da, so it was successfully established tha the Desert Chlorella GTD8A1-HLF expression systems of human lactoferrin.3. Results of the optimal product condition was showed: the best concentration of NaCO₃ 0.1mol/L、K₂HPO₄ 0.02mol/L、Na Cl 0.01mol/L、MgSO₄ 0.03mol/L、CuSO₄ 0.002mol/L、H₃BO₃ 0.02mol/L、NaNO₃ 0.6mol/L、FeSO₄ 0.006mol/L、ZnSO₄ 0.002mol/L respectively. The human lactoferrin output production reached to 51.65 mg/L.