|Title:||探討 IL-15 異構體對 IL-15 生物活性與 IL-15Rα 在細胞膜表現量的調控
Inhibition of IL-15 bioactivity and the cell surface expression of IL-15Rα by IL-15 alternatively spliced isoform
|Keywords:||介白質-15;介白質-15異構體;介白質-15受器;反轉錄病毒;細胞株;IL-15;IL-15 isoform;IL-15R;retrovirus;cell line||Issue Date:||2011||Abstract:||
Cytokines are important for the generation, regulation and maintenance of an effective immune response. Alternative splicing of pre-mRNA in IL-2 family cytokines has been reported and might play a role in regulating the function of the corresponding prototype cytokine. For example, interleukin-15 (IL-15) is a pleiotropic cytokine that mediates innate and adaptive immune responses. While expression of alternatively spliced IL-15 mRNAs is differentially distributed among tissues, detail mechanisms by which IL-15 activity is regulated by the splice variant remain unclear.
In this study, we generated a plasmid expressing IL-15 cDNA with a 48-nucleotide deletion in exon 7 (called IL-15ΔE7) from full length IL-15 cDNA by PCR method. IL-15 or IL-15ΔE7 protein obtained from the supernatant or lysates of COS-7 or 293T cells which were transiently transfected with a plasmid expressing IL-15 or IL-15ΔE7 gene at 24-48 hours was further analyzed by immunofluorescene, ELISA, Western blotting and IL-15 dependent cell proliferation assay. Expression of IL-15 and IL-15ΔE7 proteins running at around 18-20 kDa by gel electrophoresis was confirmed by Western blotting. Whereas IL-15 was mainly expressed in the cytoplasm, IL-15ΔE7 was restricted and expressed in the ER by immunofluorescent assays. Moreover, secretion of IL-15ΔE7 into the medium was significantly reduced compared to IL-15 by ELISA. Results from IL-15 dependent cell proliferation assays using HT-2 cells / MTT assay revealed that culture supernatant from cells transfected with full-length IL-15 cDNA contained bioactivity for HT-2 cells while the supernatant from COS-7 cells transfected with IL-15ΔE7 had no comparable IL-15 activity at all. Recently, we have also established an IL-15Rα stably expressing COS-7 cell line by retrovirus infection. The cell line will be used to study the binding of IL-15 and IL-15ΔE7 to IL-15Rα in future experiments.
In this study, we have established the plasmid expressing IL-15 and IL-15ΔE7 and also have characterized the differences between IL-15 and IL-15ΔE7 by transient transfection method. Results from this and the future experiments will lead to a better understanding on how IL-15 alternative splice variant could regulate IL-15 action and its impact on IL-15Rα-mediated signaling pathway. The information will be very helpful for the development of novel strategy in treating IL-15 mediated inflammatory disorders or diseases.
|Appears in Collections:||免疫學研究所|
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