Objective: To observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-Iike receptors (RLRs) signaling pathway when immunological cells were infected with HIN1. Meth...Objective: To observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-Iike receptors (RLRs) signaling pathway when immunological cells were infected with HIN1. Methods: Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN- ~, ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR). Results: The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-13 promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor K B (NF- K B) mRNA levels increased significantly (P〈0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF- K B decreased significantly in each group with significant statistic differences (P〈0.05). Conclusions: The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.展开更多
为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。...为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。经过一系列试验表明,该检测方法线性关系良好,R^(2)值为0.99;特异性强,敏感性高,最低可检测至2.23 copies/μL,比普通PCR灵敏约100倍;重复性好,组内变异系数为0.25%~0.43%,组间变异系数为0.67%~0.97%;对于各地区96份临床样品检测出PEDV阳性率为25%。本研究建立的实时荧光定量PCR检测方法为PEDV的临床诊断、流行病学调查以及定量研究提供了有效的检测工具。展开更多
Retinoic acid inducible gene-I(RIG-I)is a caspase recruitment domain(CARD)containing protein that acts as an intracellular RNA receptor and senses virus infection.After binding to double stranded RNA(dsRNA)or 5′-trip...Retinoic acid inducible gene-I(RIG-I)is a caspase recruitment domain(CARD)containing protein that acts as an intracellular RNA receptor and senses virus infection.After binding to double stranded RNA(dsRNA)or 5′-triphosphate single stranded RNA(ssRNA),RIG-I transforms into an open conformation,translocates onto mitochondria,and interacts with the downstream adaptor mitochondrial antiviral signaling(MAVS)to induce the production of type Ⅰ interferon and inflammatory factors via IRF3/7 and NF-κB pathways,respectively.Recently,accumulating evidence suggests that RIG-I could function in non-viral systems and participate in a series of biological events,such as inflammation and inflammation related diseases,cell proliferation,apoptosis and even senescence.Here we review recent advances in antiviral study of RIG-I as well as the functions of RIG-I in other fields.展开更多
试验旨在建立一种快速检测禽源沙门氏菌SYBR Green Ⅰ荧光定量PCR(qPCR)的方法,即根据沙门氏菌invA基因的保守序列设计引物,利用普通PCR方法扩增沙门氏菌invA基因保守基因片段,将其克隆到pMD18-T载体上,将获得的重组质粒pMD18-T-invA作...试验旨在建立一种快速检测禽源沙门氏菌SYBR Green Ⅰ荧光定量PCR(qPCR)的方法,即根据沙门氏菌invA基因的保守序列设计引物,利用普通PCR方法扩增沙门氏菌invA基因保守基因片段,将其克隆到pMD18-T载体上,将获得的重组质粒pMD18-T-invA作为标准阳性模板。经qPCR条件优化后,进行特异性、灵敏性和重复性试验。结果显示,所建立的SYBR Green Ⅰ qPCR方法的Ct值与标准品在1.4~1.4×10^(10)拷贝/μL范围内呈良好的线性关系,R2为0.9963,扩增效率为95%,检测下限为1.4拷贝/μL;与大肠埃希菌、金黄色葡萄球菌、链球菌、痢疾志贺菌、多杀性巴氏杆菌无交叉反应;该方法组内变异系数和组间变异系数均小于2.5%;对44份粪便样本和132份蛋液样本进行qPCR方法和常规PCR方法检测,结果显示该qPCR方法的阳性检出率分别为22.7%(10/44)、0.8%(1/132),常规PCR的阳性检出率分别为9.1%(4/44),0%(0/132)。结果表明:试验成功建立禽源沙门氏菌qPCR检测方法,可为禽源沙门氏菌的快速检测提供技术支撑。展开更多
基金Supported by the National Natural Science Foundation of China(No.81273616 and 30973693)the Doctoral Fund of the Ministryof Education 2010(No.20104401110003)the Natural Science Foundation of Guangdong Province(No.S2013010013434)
文摘Objective: To observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-Iike receptors (RLRs) signaling pathway when immunological cells were infected with HIN1. Methods: Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN- ~, ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR). Results: The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-13 promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor K B (NF- K B) mRNA levels increased significantly (P〈0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF- K B decreased significantly in each group with significant statistic differences (P〈0.05). Conclusions: The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.
文摘Retinoic acid inducible gene-I(RIG-I)is a caspase recruitment domain(CARD)containing protein that acts as an intracellular RNA receptor and senses virus infection.After binding to double stranded RNA(dsRNA)or 5′-triphosphate single stranded RNA(ssRNA),RIG-I transforms into an open conformation,translocates onto mitochondria,and interacts with the downstream adaptor mitochondrial antiviral signaling(MAVS)to induce the production of type Ⅰ interferon and inflammatory factors via IRF3/7 and NF-κB pathways,respectively.Recently,accumulating evidence suggests that RIG-I could function in non-viral systems and participate in a series of biological events,such as inflammation and inflammation related diseases,cell proliferation,apoptosis and even senescence.Here we review recent advances in antiviral study of RIG-I as well as the functions of RIG-I in other fields.
文摘试验旨在建立一种快速检测禽源沙门氏菌SYBR Green Ⅰ荧光定量PCR(qPCR)的方法,即根据沙门氏菌invA基因的保守序列设计引物,利用普通PCR方法扩增沙门氏菌invA基因保守基因片段,将其克隆到pMD18-T载体上,将获得的重组质粒pMD18-T-invA作为标准阳性模板。经qPCR条件优化后,进行特异性、灵敏性和重复性试验。结果显示,所建立的SYBR Green Ⅰ qPCR方法的Ct值与标准品在1.4~1.4×10^(10)拷贝/μL范围内呈良好的线性关系,R2为0.9963,扩增效率为95%,检测下限为1.4拷贝/μL;与大肠埃希菌、金黄色葡萄球菌、链球菌、痢疾志贺菌、多杀性巴氏杆菌无交叉反应;该方法组内变异系数和组间变异系数均小于2.5%;对44份粪便样本和132份蛋液样本进行qPCR方法和常规PCR方法检测,结果显示该qPCR方法的阳性检出率分别为22.7%(10/44)、0.8%(1/132),常规PCR的阳性检出率分别为9.1%(4/44),0%(0/132)。结果表明:试验成功建立禽源沙门氏菌qPCR检测方法,可为禽源沙门氏菌的快速检测提供技术支撑。