Cloning, Expression And Biological Function Of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL/TNFSF10) In Bat And Yangtze Finless Porpoise

This thesis contains two chapters:1.The cloning,expression,tissue distribution and biological function of bat TRAIL gene.The present study,the full-length cDNA of TRAIL(designated bTRAIL)from the bat was cloned using RT-PCR techniques,and its biological activities have been characterized.To our knowledge,this is the first report of any TRAIL gene being cloned from bat.it contained an open reading frame of 843 bp,which encoded 280 amino acid residues with a predicted protein molecular weight of 32.48 kDa.The amino acid of bTRAIL protein included a putative transmembrane domain,a TNF superfamily signature,one conserved cysteine and putative glycosylation site.Moreover,Real-time PCR suggested that bTRAIL mRNA was expressed in a tissue specific manner and primarily expressed in spleen.The Pet43.1a-bsTRAIL was efficiently expressed in Escherichia coli BL21(DE3)and confirmed by SDS-PAGE and Western blot analysis.In vitro,the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide(MTT)assay?TrypanBlue and Flow Cytometry analysis indicated that the purified soluble TRAIL was able to induce apoptosis of Jurkat and HeLa cells in a dose-dependent manner.Our work may provide the basis investigations of bat innate immunity.These results about bTRAIL gene would provide a basis for understanding the characteristics of adaptive immune and innate immunity in the bat.2.The cloning,expression and biological function of the finless porpoises TRAIL gene.The full-length cDNA of TRAIL(designated fTRAIL)from the finless porpoises was cloned using RT-PCR techniques,and its biological activities have been characterized.It contained an open reading frame of 864 bp,which encoded 288 amino acid.This protein was found to contain a predicted transmembrane domain,putative glycosylation site,one conserved cysteine,and a typical TNF homology domain corresponding to other known TRAIL homologues.Sequence comparison indicated that the amino acid sequence of fTRAIL was very similar to its horse(86%),human(79%),dog(82%)and cow(80%)counterparts.Recombinant soluble fTRAIL(pET43.1a-fsTRAIL)fused with Nus-his6 tag was efficiently expressed in Escherichia coli BL21(DE3).Phylogenetic analysis indicated the finless porpoises have a closely relationship with artiodactyla.the purified soluble TRAIL was able to induce apoptosis of Jurkat and HeLa cells in a dose-dependent manner.The present findings may provide valuable information for research into the immune system of the finless porpoise.