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目的:研究钙调神经磷酸酶(CaN)与活化T细胞核因子(NFAT)相互作用保守位点衍生的多肽和人免疫缺陷病毒(HIV)TAT的融合多肽对CaN-NFAT信号途径的影响。方法:根据CaN-NFAT的结合位点经计算机优化后得到PVIGIT,并和TAT融合成新的多肽PepNFAT-TAT,用异硫氰酸荧光素(FITC)荧光标记后加至携带CaN催化亚基cDNA的重组质粒pRED-CnA转染过的神经细胞LN-229中,再用离子霉素刺激,用激光共聚焦显微镜观察该融合多肽的细胞定位,并检测该多肽对CaN酶活性的影响,报告基因pNFAT-luc实验检测其对NTAF激活的影响,实时定量RT-PCR检测其对NFAT下游基因白细胞介素-4(IL-4)表达的影响。结果:该融合蛋白定位于细胞质中,报告基因实验显示该多肽能够抑制对NFAT的激活,抑制离子霉素诱导IL-4的表达。结论:融合多肽PepNFAT-TAT能抑制CaN-NFAT信号途径,但对CaN的酶活性没有影响,是潜在的免疫抑制剂。
AIM: To investigate the effect of fusion polypeptide derived from calcineurin (CaN) and activated T cell nuclear factor (NFAT) -conjugated site fusion protein and human immunodeficiency virus (HIV) TAT on CaN-NFAT signal pathway. METHODS: PVIGIT was obtained by computer optimization based on the binding site of CaN-NFAT. Peptide-TAT was fused with TAT and fluorescently labeled with fluorescein isothiocyanate (FITC) and then added to CaN-catalyzed subunit cDNA The recombinant plasmid pRED-CnA was transfected into LN-229 cells. The cells were stimulated with ionomycin, and the localization of the fusion polypeptide was detected by laser confocal microscopy. The effect of the polypeptide on the activity of CaN was detected. The reporter gene The effect of pNFAT-luc on the activation of NTAF was detected by real-time RT-PCR. The expression of interleukin-4 (IL-4), a downstream NFAT gene, was detected by real-time quantitative RT-PCR. Results: The fusion protein located in the cytoplasm, the reporter gene experiments showed that the polypeptide can inhibit NFAT activation, inhibition of ionomycin-induced IL-4 expression. Conclusion: The fusion peptide PepNFAT-TAT can inhibit the CaN-NFAT signaling pathway, but has no effect on the enzymatic activity of CaN and is a potential immunosuppressant.