ABSTRACT
Objective To construct the recombinant adenovirus encoding human p16 gene for future gene therapy.Methods The human p16 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria.Then the recombinant adenovirus was transfected into 293T cells using Lipofectine DOTAP.The target gene was detected by polymerase chain reaction(PCR).The titer and its infection rate were determined using the green fluorescent protein(GFP) expression in the shuttle plasmid.Results Restriction endonuclease and PCR analysis confirmed that the human p16 gene was successfully inserted into the adenovirus vector.The titer of the recombinant adenovirus was 6.1?10~(10)pfu/ml.The adenovirus has a strong effect on human fibroblast cells.Conclusion The recombinant adenovirus containing human p16 gene was successfully constructed by the method of homogenous recombination in bacteria.
ABSTRACT
Objective To construct the recombinant adenovirus encoding human IL-24 gene for future gene therapy. Methods The human IL-24 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into 293T cells using Lipofectine DOTAP. The target gene was detected by polymerase chain reaction (PCR). The titer and its infection rate were determined using the green fluorescent protein (GFP) expression in the shuttle plasmid. The expression of target protein was measured by the method of immunohistochemistry. Results Restriction endonuclease and PCR analysis confirmed that the human IL-24 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.2?10 10 pfu/ml. The adenovirus has a strong effect on A549 cells and human IL-24 can express in it. Conclusion The recombinant adenovirus containing human IL-24 gene was successfully constructed by the method of homogenous recombination in bacteria.
ABSTRACT
Objective To investigate the fermentation conditions for the heat-labile enterotoxin B subunit (LTB) of genetic recombinant E.coli. Methods The effects of different kinds of media, the range of pH, the induction time, the glucose concentration, and the fed-batch on LTB level in 10-liter fermenter under the condition of cascade controlling dissolved oxygen content were analyzed. Results Under the established conditions, the cell output per liter was 40.5g, and the expression level was 30.6%. Conclusion The results indicate that the stable fermentation technology with high efficiency has been established.