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

吳秉峰(Ping-Feng Wu)

指導教授 ： 顧家綺

國立臺灣大學/醫學院/免疫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.03356

摘要

無資料

關鍵字

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

並列摘要

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.

並列關鍵字

IL-15 ； IL-15 isoform ； IL-15R ； retrovirus ； cell line

參考文獻

2. Anderson, D.M., Kumaki, S., Ahdieh, M., Bertles, J., Tometsko, M., Loomis, A., Giri, J., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., et al. (1995b). Functional characterization of the human interleukin-15 receptor alpha chain and close linkage of IL15RA and IL2RA genes. J Biol Chem 270, 29862-29869.

3. Angiolillo, A.L., Kanegane, H., Sgadari, C., Reaman, G.H., and Tosato, G. (1997). Interleukin-15 promotes angiogenesis in vivo. Biochem Biophys Res Commun 233, 231-237.

4. Azimi, N., Brown, K., Bamford, R.N., Tagaya, Y., Siebenlist, U., and Waldmann, T.A. (1998). Human T cell lymphotropic virus type I Tax protein trans-activates interleukin 15 gene transcription through an NF-kappaB site. Proc Natl Acad Sci U S A 95, 2452-2457.

5. Bamford, R.N., Battiata, A.P., Burton, J.D., Sharma, H., and Waldmann, T.A. (1996a). Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I region /IL-15 fusion message that lacks many upstream AUGs that normally attenuates IL-15 mRNA translation. Proc Natl Acad Sci U S A 93, 2897-2902.

6. Bamford, R.N., Battiata, A.P., and Waldmann, T.A. (1996b). IL-15: the role of translational regulation in their expression. J Leukoc Biol 59, 476-480.