【摘要】 第10号染色体上缺失的磷酸酶与张力蛋白同源物基因(phosphatase and tensin homolog deleted on chromosome ten, PTEN)是迄今发现的第1个具有双特异性磷酸酶活性的抑癌基因，在多种信号通路中发挥重要作用。为了阐明鱼类PTEN在核因子κB (nuclear factor kappa-B, NF-κB)、干扰素(interferon,IFN)和激活蛋白-1 (activator protein-1, AP-1)信号通路中的调控作用，本研究利用逆转录PCR (reverse transcription PCR, RT-PCR)方法从大口黑鲈(Micropterus salmoides)中扩增并克隆了PTEN基因ORF序列，利用qPCR检测了不同病原体相关分子模式—诺卡氏菌(Nocardia seriolae)、大口黑鲈蛙病毒(Largemouth bass virus, LMBV)及聚胞肌酸Poly I:C对大口黑鲈PTEN基因表达水平变化的影响。通过构建真核表达质粒pCMV-Flag-PTEN对大口黑鲈PTEN进行亚细胞定位以及过表达对NF-κB、AP-1、IFN启动子荧光素酶活性的激活作用进行研究。蛋白质序列分析显示，大口黑鲈PTEN编码了439个氨基酸，其中包含1个PTPc结构域和1个PTEN＿C2结构域，在系统发育树中与其他硬骨鱼类PTEN家族聚为一支。qPCR检测发现PTEN在健康的大口黑鲈多种组织中广泛表达，且在脾脏和鳃中高表达。人工感染诺卡氏菌可引起PTEN基因表达量显著下降；注射LMBV、Poly I:C后，PTEN基因表达量呈先下降后上升或先上升后下降的表达趋势。亚细胞定位结果显示，大口黑鲈PTEN在HEK-293T细胞的细胞质和细胞核中分布。双荧光素酶活性分析发现过表达pCMV-Flag-PTEN可以增强AP-1和抑制NF-κB、IFN启动子荧光素酶活性。以上研究表明，大口黑鲈PTEN可以通过激活AP-1和抑制NF-κB、IFN信号通路，在机体抗细菌和抗病毒先天免疫应答中发挥重要的调控作用。本研究为阐明大口黑鲈抗病免疫应答机制的研究提供基础资料，为抗菌疫苗开发、抗病品种(系)的选育提供理论基础。更多还原

【Abstract】 Phosphatase and tensin homolog deleted on chromosome ten(PTEN) is the first tumor suppressor gene with bispecific phosphatase activity, which plays an important role in many signaling pathways. To clarify the regulatory role of PTEN in nuclear factor kappa-B(NF-κB), interferon(IFN) and activator protein-1(AP-1) signaling pathways, which are closely related to the immune response in fish, in this study, the ORF region of PTEN gene was cloned from largemouth bass(Micropterus salmoides), the effects of pathogenassociated molecular patterns Nocardia seriolae, Largemouth bass virus(LMBV), Poly I:C challenge on PTEN gene expression in largemouth bass were detected by qPCR in vivo. To investigate the subcellular localization of PTEN, eukaryotic expression plasmid p CMV-Flag-PTEN was constructed, and the effect of PTEN overexpression on NF-κB, AP-1 and IFN promoter activation was investigated. Protein sequence analysis showed that PTEN encoded a 439-amino acid protein, including a PTPc domain and a PTEN＿C2 domain. In the phylogenetic tree, the PTEN protein of largemouth bass was clustered with other fish. The results of qPCR showed that PTEN was widely expressed in many tissues, and highly expressed in spleen and gill. After LMBV and Poly I:C stimulation, the expression of PTEN decreased first and then increased or increased first and then decreased. The results of subcellular localization showed that PTEN was distributed in the cytoplasm and nucleus of HEK-293T cells. Double luciferase activity assay showed that overexpression of PTEN could enhance AP-1 activation and inhibit the activity of NF-κB and IFN promoter. These results suggested that PTEN played an important regulatory role in the innate immune response against bacteria and viruses by activating AP-1 and inhibiting NF-κB and IFN signaling pathways. This study provides basic data for the clarify of immune response mechanism of largemouth bass against disease, and provides theoretical basis for the development of antibacterial vaccine and the breeding of disease-resistant varieties(lines).更多还原