| X-chromosomal linked inhibitor of apoptosis（XIAP）is the strongest anti apoptotic factor in the IAP family and has the widest range of action.It can bind to Caspase and inhibit its activity to inhibit cell apoptosis.XIAP associated factor 1（XAF1）is an interacting protein of XIAP that can bind to and inhibit its effects.This article characterized the XAF1 and XIAP of grass carp and analyzed their expression patterns and functions.The main research results are as follows:The XAF1 and XIAP genes of grass carp（Ctenopharyngodon idella）were cloned and sequenced.The open reading frame of XAF1 was 789 bp encoding 262 aa with a protein molecular weight of approximately 31.5 KD.The open reading frame of XIAP was 1371 bp encoding 456 aa with a protein molecular weight of approximately 51.5KD.Homologous sequence analysis showed that XAF1 contained seven zinc finger,including six C2HC zinc finger motifs and one C2H2 zinc finger motif;XIAP contains three BIR domains arranged in the CX2CX16HX6C pattern,one Ring domain with E3ubiquitin ligase activity,and one UBA domain.Among them,grass carp XAF1 and XIAP have the highest sequence consistency with zebrafish XAF1（71%）and XIAP（64%）,respectively.The phylogenetic tree also indicates that grass carp and zebrafish have the closest genetic relationship.Real time fluorescence quantitative PCR results showed that XAF1 and XIAP were constitutively expressed in all examined tissues/organs.Among them,XAF1 had the highest expression level in the blood and XIAP had the highest expression level in the heart.In vitro isolated peripheral blood lymphocytes（PBL）of grass carp,the XAF1transcription level was upregulated by 8.6-fold,and the XIAP transcription level was upregulated by 3.7-fold at 6 h after treatment with IFN-γ.Grass carp reovirus（GCRV）slightly upregulated the transcription level of XAF1 in grass carp kidney cells,but did not affect the transcription level of XIAP.Through stable transfection and screening,ptGFP1XAF1 cell lines and ptGFP1XIAP cell lines overexpressing XAF1 and XIAP respectively were established.The proliferation experiment results showed that overexpression of XAF1 and XIAP did not affect the growth and proliferation rate of CIK.Overexpression of XAF1 can reduce the infectivity of GCRV to CIK,while overexpression of XIAP can enhance the infectivity of GCRV to CIK.The half lethal concentrations of pt GFP1 cell line,pt GFP1XAF1 cell line and pt GFP1XIAP cell line to GCRV are 10-6.62/m L,10-5.86/m L and 10-6.89/m L,respectively.Overexpression of XAF1 increases the sensitivity of CIK to cisplatin,but overexpression of XIAP does not affect the sensitivity of CIK to cisplatin.The half lethal concentrations of cisplatin in the pt GFP1 cell line,pt GFP1XAF1 cell line,and pt GFP1XIAP cell line are 30.86μM,22.09μM and 30.5μM respectively. |
