Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition · 2015
[Construction of Recombinant Adenovirus Vector with Human/Mouse Interferon Gene and Its Transfection into Human Umbilical Cord Mesenchymal Stem Cells].
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Li Hui, Hu Qiong-ying, Jia Yong-qian
Аннотация
OBJECTIVE: Construction and identification of recombinant adenovirus vector with human/mouse interferon (IFN-gamma) and effectively transfection into human umbilical cord mesenchymal stem cells (HUMSCs).
METHODS: IFN-gamma gene of human/mouse were amplified from the plasmid by polymerase chain reaction (PCR) and then inserted into the plasmid pDC316 to generate pDC316-IFN-gamma. After being confirmed by restriction enzyme digestion and DNA sequencing, the DNA encoding IFN-gamma in the new structure were inserted into the vector of recombinant plasmid adenovirus and confirmed by restricition enzyme digestion. Then the human embryonic kidney cell line 293 were transfected with confirmed Ad-IFN-gamma, and the recombinant adenovirus were amplified, and the virus titer were detected using 50% tissue culture infective dose (TCID50) assay. The expression of the green fluorescent protein (GFP) and IFN-gamma were detected by fluorescent microsope and Western blot and ELISA after the recombinant adenovirus transfected HUMSCs.
RESULTS: The recombinant adenovirus Ad-hIFN-gamma were constructed successfully, and amplified with titer of 1.6 x 10(10) IU/mL. The titer of Ad-mIFN-gamma was 1.0 x 10(10) IU/mL and the titer of Ad-GFP was 1.0 x 10(9) IU/mL. The green fluorescence proteins could be observed under fluorescent microscope in HUMSCs 24 h after transfection and with a stronger degree after 72 h, and IFN-gamma expression in HUMSCs were confirmed by Western blot and ELISA.
CONCLUSION: Construction and identification of recombinant adenovirus vector of human/mouse IFN-ygamma and effectively transfection of HUMSCs were successful.
METHODS: IFN-gamma gene of human/mouse were amplified from the plasmid by polymerase chain reaction (PCR) and then inserted into the plasmid pDC316 to generate pDC316-IFN-gamma. After being confirmed by restriction enzyme digestion and DNA sequencing, the DNA encoding IFN-gamma in the new structure were inserted into the vector of recombinant plasmid adenovirus and confirmed by restricition enzyme digestion. Then the human embryonic kidney cell line 293 were transfected with confirmed Ad-IFN-gamma, and the recombinant adenovirus were amplified, and the virus titer were detected using 50% tissue culture infective dose (TCID50) assay. The expression of the green fluorescent protein (GFP) and IFN-gamma were detected by fluorescent microsope and Western blot and ELISA after the recombinant adenovirus transfected HUMSCs.
RESULTS: The recombinant adenovirus Ad-hIFN-gamma were constructed successfully, and amplified with titer of 1.6 x 10(10) IU/mL. The titer of Ad-mIFN-gamma was 1.0 x 10(10) IU/mL and the titer of Ad-GFP was 1.0 x 10(9) IU/mL. The green fluorescence proteins could be observed under fluorescent microscope in HUMSCs 24 h after transfection and with a stronger degree after 72 h, and IFN-gamma expression in HUMSCs were confirmed by Western blot and ELISA.
CONCLUSION: Construction and identification of recombinant adenovirus vector of human/mouse IFN-ygamma and effectively transfection of HUMSCs were successful.