为表达重组牛干扰素ω12(rBoIFN-ω12),并分析该蛋白的稳定性和免疫调节机制,本研究首先从牛的肝脏基因组中扩增牛IFN-ω12基因,构建克隆载体,从克隆载体中扩增牛IFN-ω12成熟肽编码基因,并将其克隆于p ET-32a载体构建了重组表达载体p E...为表达重组牛干扰素ω12(rBoIFN-ω12),并分析该蛋白的稳定性和免疫调节机制,本研究首先从牛的肝脏基因组中扩增牛IFN-ω12基因,构建克隆载体,从克隆载体中扩增牛IFN-ω12成熟肽编码基因,并将其克隆于p ET-32a载体构建了重组表达载体p ET-32a-Bo IFNω12,经酶切和测序鉴定正确后转化大肠杆菌诱导表达重组蛋白r Bo IFNω12。在不同细胞系中检测纯化后r Bo IFNω12蛋白的生物学活性,结果显示r Bo IFNω12在MDBK和PK-15细胞中对水泡性口炎病毒(VSV)具有抗病毒活性;此外,40μg/m L的r Bo IFNω12在MDBK细胞中相对r Bo IFNαA有较低细胞毒性。经酸碱或高温处理后,r Bo IFNω12仍具有抗病毒活性。通过双荧光素酶试验在牛肺细胞(BL)中检测r Bo IFNω12蛋白对IFN信号通路中关键元件的影响,结果显示与未加r Bo IFN-ω12的对照细胞相比,r Bo IFNω12能够有效激活NF-κB响应元件、ISRE结合元件和Bo IFN-β启动子调控的荧光素酶活性。通过荧光定量PCR和western blot检测r Bo IFNω12作用MDBK细胞后不同时间IFN刺激因子(ISG)的表达情况,结果显示r Bo IFNω12作用MDBK细胞后,细胞中ISG15、ISG56、Mx-1的m RNA转录水平及表达量在3 h~24 h逐渐增多,Mx-1蛋白表达量在6 h~24 h比未处理组增多。本研究表明牛IFN-ω12具有抗病毒和抗细胞增殖活性,细胞毒性较低,可刺激细胞中NF-κB下游ISG中的Mx1、ISG15、ISG56的表达,并能启动细胞中NF-κB、ISRE、Bo IFN-β启动子激活的荧光素酶活性,具有典型I型IFN的特征,可作为有效的抗病毒药物,为牛IFN-ω系统的进一步研究提供思路。展开更多
AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in...AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in Huh7 cells using pc DNA3.1(+) vector. The expression of mi R-93-5 p and interferon receptor 1(IFNAR1) was measured using quantitative reverse transcriptionpolymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of mi R-93-5 p and IFNAR1 were performed using mi R-93-5 p agomir and antagomir, and pc DNA3.1-IFNAR1 and IFNAR1 si RNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of mi R-93-5 p. Cellular experiments were also conducted.RESULTS Serum mi R-93-5 p level was increased in patients with HCV-1 b infection and decreased to normal level after HCV-1 b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum mi R-93-5 p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1 b core protein increased mi R-93-5 p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of mi R-93-5 p, and IFNAR1 restore could rescue mi R-93-5 p-reduced STAT1 phosphorylation, suggesting that the mi R-93-5 p-IFNAR1 axis regulates the IFN signaling pathway.CONCLUSION HCV-1 b core protein-induced mi R-93-5 p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the mi R-93-5 p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1 b infection.展开更多
Objective:To determine the relationship of the capacity to produce interferon gamma(IFN-γ) in whole blood,bacteriological,hematological,radiographic and clinical presentations in new,HIV seronegative cases of pulmona...Objective:To determine the relationship of the capacity to produce interferon gamma(IFN-γ) in whole blood,bacteriological,hematological,radiographic and clinical presentations in new,HIV seronegative cases of pulmonary tuberculosis(TB).Methods:80 cases and 50 control subjects aged 15 years onwards,representative of Kasturba Hospital and Nursing schools of Wardha district of Maharashtra state in India were examined for their health condition with standard methodology.Results:Among these TB patients,73.8%were Quantiferon-TB gold (QFT) positive with IFN-γconcentration as 0.35 IU or more and there was none in healthy controls.The mean IFN-γconcentrations varied between 9.58 IU(50-59 yrs) and 2.58 IU(≥60 yrs),showing no trend.The differences in positivity and mean IFN-γconcentrations were statistically insignificant.Both the QFT positivity and IFN-γconcentrations were higher in normal lymphocyte percent as compared to below and above normal,but differences were not statistically significant.Conclusions:The IFN-γconcentrations are not correlated with any of the predictors of disease severity studied,the levels are significantly higher in observation group as compared to healthy group.展开更多
Multiple sclerosis（MS）is a chronic autoimmune disease of the central nervous system（CNS）characterized by coexisting processes of inflammation,demyelination,axonal neurodegeneration,and gliosis.It is the most commo...Multiple sclerosis（MS）is a chronic autoimmune disease of the central nervous system（CNS）characterized by coexisting processes of inflammation,demyelination,axonal neurodegeneration,and gliosis.It is the most common disabling neurological disease in young adulthood.展开更多
文摘为表达重组牛干扰素ω12(rBoIFN-ω12),并分析该蛋白的稳定性和免疫调节机制,本研究首先从牛的肝脏基因组中扩增牛IFN-ω12基因,构建克隆载体,从克隆载体中扩增牛IFN-ω12成熟肽编码基因,并将其克隆于p ET-32a载体构建了重组表达载体p ET-32a-Bo IFNω12,经酶切和测序鉴定正确后转化大肠杆菌诱导表达重组蛋白r Bo IFNω12。在不同细胞系中检测纯化后r Bo IFNω12蛋白的生物学活性,结果显示r Bo IFNω12在MDBK和PK-15细胞中对水泡性口炎病毒(VSV)具有抗病毒活性;此外,40μg/m L的r Bo IFNω12在MDBK细胞中相对r Bo IFNαA有较低细胞毒性。经酸碱或高温处理后,r Bo IFNω12仍具有抗病毒活性。通过双荧光素酶试验在牛肺细胞(BL)中检测r Bo IFNω12蛋白对IFN信号通路中关键元件的影响,结果显示与未加r Bo IFN-ω12的对照细胞相比,r Bo IFNω12能够有效激活NF-κB响应元件、ISRE结合元件和Bo IFN-β启动子调控的荧光素酶活性。通过荧光定量PCR和western blot检测r Bo IFNω12作用MDBK细胞后不同时间IFN刺激因子(ISG)的表达情况,结果显示r Bo IFNω12作用MDBK细胞后,细胞中ISG15、ISG56、Mx-1的m RNA转录水平及表达量在3 h~24 h逐渐增多,Mx-1蛋白表达量在6 h~24 h比未处理组增多。本研究表明牛IFN-ω12具有抗病毒和抗细胞增殖活性,细胞毒性较低,可刺激细胞中NF-κB下游ISG中的Mx1、ISG15、ISG56的表达,并能启动细胞中NF-κB、ISRE、Bo IFN-β启动子激活的荧光素酶活性,具有典型I型IFN的特征,可作为有效的抗病毒药物,为牛IFN-ω系统的进一步研究提供思路。
基金Supported by National Natural Science Foundation of China,No.81371849the TMMU Key Project for Clinical Research,No.2012XLC05
文摘AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in Huh7 cells using pc DNA3.1(+) vector. The expression of mi R-93-5 p and interferon receptor 1(IFNAR1) was measured using quantitative reverse transcriptionpolymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of mi R-93-5 p and IFNAR1 were performed using mi R-93-5 p agomir and antagomir, and pc DNA3.1-IFNAR1 and IFNAR1 si RNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of mi R-93-5 p. Cellular experiments were also conducted.RESULTS Serum mi R-93-5 p level was increased in patients with HCV-1 b infection and decreased to normal level after HCV-1 b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum mi R-93-5 p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1 b core protein increased mi R-93-5 p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of mi R-93-5 p, and IFNAR1 restore could rescue mi R-93-5 p-reduced STAT1 phosphorylation, suggesting that the mi R-93-5 p-IFNAR1 axis regulates the IFN signaling pathway.CONCLUSION HCV-1 b core protein-induced mi R-93-5 p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the mi R-93-5 p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1 b infection.
文摘Objective:To determine the relationship of the capacity to produce interferon gamma(IFN-γ) in whole blood,bacteriological,hematological,radiographic and clinical presentations in new,HIV seronegative cases of pulmonary tuberculosis(TB).Methods:80 cases and 50 control subjects aged 15 years onwards,representative of Kasturba Hospital and Nursing schools of Wardha district of Maharashtra state in India were examined for their health condition with standard methodology.Results:Among these TB patients,73.8%were Quantiferon-TB gold (QFT) positive with IFN-γconcentration as 0.35 IU or more and there was none in healthy controls.The mean IFN-γconcentrations varied between 9.58 IU(50-59 yrs) and 2.58 IU(≥60 yrs),showing no trend.The differences in positivity and mean IFN-γconcentrations were statistically insignificant.Both the QFT positivity and IFN-γconcentrations were higher in normal lymphocyte percent as compared to below and above normal,but differences were not statistically significant.Conclusions:The IFN-γconcentrations are not correlated with any of the predictors of disease severity studied,the levels are significantly higher in observation group as compared to healthy group.
基金Dr.Mao-Draayer has served as a consultant and/or received grant support from:Acorda,Bayer Pharmaceutical,Biogen Idec,EMD Serono,Genzyme,Novartis,Questor,Teva Neuroscience and Chugai PharmaDr.Mao-Draayeris currently supported by grants from NIH NIAID Autoimmune Center of Excellence:UM1-AI110557+1 种基金NIH NINDS R01-NS080821the University of Michigan Neurology Department
文摘Multiple sclerosis（MS）is a chronic autoimmune disease of the central nervous system（CNS）characterized by coexisting processes of inflammation,demyelination,axonal neurodegeneration,and gliosis.It is the most common disabling neurological disease in young adulthood.
