ABSTRACT
Objective To construct the recombinant adenovirus of mouse Osf2/Cbfal gene and to observe its ability to infect NIH3T3 fibroblasts. Methods The Osf2/Cbfal 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 NIH3T3 cells using Lipofectine DOTAP, The target gene was detected by poly-merase 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 analyses confirmed that the Osf2/Cbfal gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.6?1012 pfu/ml. The adenovirus had a strong effect on NIH3T3 cells. Conclusion The recombinant adenovirus containing Osf2/Cbfal gene was successfully constructed by the method of homogenous recombination in bacteria.
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
Monascus spp.,a kind of filamentous fungi,produce abundant of important metabolites which were widely used in the fields of food and medicine.Until now,there are few reports on the important functional genes of the Monascus spp.due to little genetic information.In this paper,the feasibility of gene deletion mediated via Agrobacterium tumefaciens on the basis of homologous recombination was analyzed by studying on the deletion of the RGS domain of putative G-protein signaling regulator gene mrfA in Monascus ruber.The length of homologous arms of deletion vector pC805S were 958 bp and 824 bp,respectively.There were 26 transformants in which homologous recombination occurred in 138 transformants and the recombination rate was 18.8%.The result showed it was feasible to identify the function of unknown gene in M.ruber with the targeted-deletion technology mediated via A.tumefaciens.