Construction Of Recombinant For Adenovirus Expression Vector Encoding IKK2dn Gene And Identification Of Their Expression

Objective: (1) To construct recombinant eukaryotic expression adenovirus vector pAdxsi-GFP-IKK2dn; (2)To pack the adenovirus vector, to cotransfect with mammalian cell such as hela and to express in hela in order to study the efficiency of transgene IKK2dn on inducing immunetolerance, in order to protect allograft in organic transplantation.Methods: (1) After being digested by KpnI/HindIII, IKK2dncDNA and adenovirus vector pShuttle-GFP-CMV(-)TEMP were purified by low meiting temperature agarose and then ligated by T4DNA ligase. Products of ligation were transformed to competent cells DH5a and the transformed cells were selected by Amikacin. Colony were used for digestion analysis. Plasmids of pShuttle-GFP-CMV(-)TEMP-IKK2dn isolated from positive transformed clones. (2) After being digested by I-CeuI+I-SceI, pShuttle-GFP-CMV(-)TEMP-IKK2dn and pAdxsi were purified by low meiting temperature agarose and then ligated by T4DNA ligase. Products of ligation were transformed to competent cells DH5a and the transformed cells were selected by ampicillin. Colony were used for digestion analysis. Plasmids of pAdxsi-GFP-IKK2dn isolated from positive transformed clones were made preparation for packaging of the adenovirus vector. (3) Human embroic kidney 293T cells were cotransfected with pAdxsi-GFP-IKK2dn and Lipofectamine2000. After observing the coming out of adenovirus, then harvested the adenovirus, and then freezed, intreased and rereceived the adenovirus, we centrifugaled, dialyzed and puried the adenovirus for its titre determination. (4) Cotransfect the adenovirus with hela cell and to express in hela in order to study the efficiency of transgene IKK2dn.Resules: (1) A segment of 7.4kbp for Shuttle-GFP-CMV(-)TEMP-IKK2dn was obtained. (2) After extracting out recombinant plasmid which is isolated from positive transformed clones, the sequence of cloned rat IKK2dn gene was proved to be correct. (3) The virus titre of 2x10¹¹ PFU/ml is sufficient for further research. (4) And use the RT-PCR method to identify their expressions to be reliable and efficient.Conclusions: (1) Shuttle-GFP-CMV(-)TEMP-IKK2dn fragment was successfully cloned. (2) The recombinant eukaryotic expression adenovirus vector pAdxsi-GFP-IKK2dn was constructed successfully.(3) pAdxsi-GFP-IKK2dn and Lipofectamine2000 were successfully packaged into adenovirus particle with high titre and good stability.(4) The adenovirus was constructed with hela cell successfully and those results can be used for further research of transgenic inducing immunologic tolerance and protecing allgrafts in organ transplantation.