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Construction of Recombinant Adenovirus Vector of Indoleamine 2,3-dioxygenase and Chimeric Albumin Promoter

Author: BaiYanNan
Tutor: WangYaoDong
School: Fujian Medical
Course: Surgery
Keywords: IDO Albumin promoter Adenovirus
CLC: R392.4
Type: Master's thesis
Year: 2009
Downloads: 47
Quote: 1
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Abstract


[Objective] To construct to albumin promoter promoter and IDO gene recombinant adenovirus vector carrying mice , the researchers in the level of transcription and protein expression in the liver Hepa 1-6 cells . [Methods] in accordance with the basic principles of design primer design albumin promoter primers , synthetic albumin promoter , subcloned into adenovirus promoter shuttle vector pAdTrack XbaI / HindIII restriction sites to construct carrying the murine pAdTrack-PmALB carrier protein promoter ; digestion containing the mouse full-length IDOcDNA PCOUS-2 plasmid and subcloned into the shuttle vectors pAdTrack- PmALB pAdTrack- CMV XhoI / EcoRV restriction sites , in BJ5183 bacteria and homologous recombination AdEasy-1 , generating and screening adenovirus containing the gene skeleton the plasmid pAdEasy-CMV/mIDO pAdEasy-malb/mIDO positive clones , digested , correct sequencing , the liposomes were transfected with AD- 293 cells for packaging , amplified , detection of virus titer , the identification and detection of the RT - PCR and fluorescence microscope adenovirus AD-293 cells transfected IDO expression . The will containing the gene mIDO , virus Hepa 1-6 cells transfected RT-PCR and Western Blot analysis of gene and protein expression were detected Hepa 1-6 cells mIDO . 【Results】 carry the albumin promoter and IDO gene recombinant adenovirus vector was constructed successfully confirmed by restriction enzyme digestion and sequencing , RT-PCR was used to detect the expression of IDO AD-293 transfected cells , the virus titer of 2.9 × 106 pfu / ml and can infect Hepa 1-6 cells can be detected by RT- PCR and Western Blot IDO expression in Hepa 1-6 cells . [Conclusion] successfully constructed a recombinant adenovirus vector carrying albumin promoter and mouse IDO gene can infect Hepa 1-6 cells at the level of transcription and protein expression , and laid the foundation to explore the biological function of IDO .

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