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- W2368972669 abstract "AIM: To construct PSNAV2.0-tumor necrosis factor α(TNFα) recombined plasmid that can continuously secret human (hTNF-α) in human embryo kidney cells (HEK-293),and detect its transient protein expression in vitro,in order to lay a foundation with this gene for microencapsulated cell transplantation and improving diseases. METHODS: The experiment was conducted in the Cytobiology Laboratory,Institute of Gerontology,General Hospital of Chinese PLA,from June 2006 to May 2007. ①cDNA sequence with hTNF-α was used as template to gain hTNF-α gene fragment by PCR,which was inserted directionally into eukaryotic expression vector PSNAV2.0,thus recombined plasmid PSNAV2.0-TNF α was attained. Through double enzyme incision by Sal Ⅰ and EcoR Ⅰ,PCR and sequence detection of inserted fragment,the plasmid was characterized. ②The plasmid was then transfected into human HEK-293 cells by using cationic liposome to construct the gene engineering cells that could secret continuously hTNF-α. RT-PCR and Western blot were used to detect the transient expression of hTNF α protein in vitro in the supernatant of transfected cells. RESULTS: ①Through double enzyme incision by Sal Ⅰ and EcoR Ⅰ,PCR and sequence detection,the inserted fragment was proved correct. ②Results of RT-PCR and Western blot also showed that hTNF α (Mr 17 000) existed in the supernatant of cultured HEK-293 cells. CONCLUSION: A eukaryotic expression vector of recombined plasmid PSNAV2.0-TNF α is successfully constructed,and hTNF-α is effectively secreted outside the cells after its transfection into HEK-293 cells." @default.
- W2368972669 created "2016-06-24" @default.
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- W2368972669 date "2007-01-01" @default.
- W2368972669 modified "2023-09-26" @default.
- W2368972669 title "Construction and transient protein expression of PSNAV2.0-TNF alfa recombined plasmids in vitro" @default.
- W2368972669 hasPublicationYear "2007" @default.
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