Recombination And Identification Of PPARγ Gene Interference Adenovirs Vector

Objective: To construct PPARγ gene interference adenoviral vector for lay the foundation of investigation of the functions and elucidation of the molecular mechanisms of PPARγ gene in traumatic brain injury.Methods: Four siRNAs be designed for human PPAR gamma gene, siRNAs were transfected into LN229 cells by liposome transfection method. RT-PCR be used to detect gene interference effect. Choose the siRNA with the best interferential effect and using artificial synthesized to compound shRNA that with restriction sites of Sfi at both ends according to its secquences. Used Sfi to digest pAd-pSES-HUS plasmid, T4 ligase be used to connect shRNA to pAd-PSES-HUS plasmid. Verification of recombinant plasmid with double enzyme digestion by Pmel and EcoRV. Using Pmel to digest recombinant plasmid and then transferred it in bacterial strain BJ5183 and homologous recombination adenovirus plasmid: pAd-PSES-HUS plasmid and adenovirus backbone plasmid(p Adeasy1). Verification of homologous recombination adenovirus plasmid by Pacl enzyme digestion and transformed it into HEK293 cells for pack adenovirus. Virus titer be test after obtain the adenovirus, The interference effect of recombinant adenovirus be verify by RT-PCR and Western-blot.Results:(1) The pAd-pSES-HUS-PPARγ-shRNA shuttle plasmid was successfully constructed.(2) Obtained the homologous recombination adenovirus vector with high efficiency of interference.(3) The recombinant adenovirus vector with interfering PPAR gamma gene was successfully constructed.Conclusion: In this study, we successfully constructed the recombinant adenovirus vector with interfering PPAR gamma gene, To lay the foundation for the study of the function of PPAR gamma gene in the inflammatory reaction.