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。
