甲型流感病毒(Influenza A virus,IAV)是引起每年季节性流感及偶尔的流感大流行的主要病原体,严重危害公共卫生和社会经济。IAV属于分节段的单股负链RNA病毒,其基因组编码多种不同的蛋白质,其中非结构蛋白1(non-structural protein 1,N...甲型流感病毒(Influenza A virus,IAV)是引起每年季节性流感及偶尔的流感大流行的主要病原体,严重危害公共卫生和社会经济。IAV属于分节段的单股负链RNA病毒,其基因组编码多种不同的蛋白质,其中非结构蛋白1(non-structural protein 1,NS1)是IAV的调节宿主免疫应答和病毒致病性的重要毒力因子。NS1具有多种功能,它可以与多种宿主RNA及细胞因子相互作用,在增强病毒mRNA翻译、抑制I型干扰素和细胞凋亡等方面发挥重要作用,因此,深入研究IAV NS1在宿主细胞中的作用,对于更好地理解病毒感染和复制的机制是至关重要的。论文主要根据NS1的免疫逃逸机制、促进病毒复制和适应性进化,以及NS1作为新型治疗靶点在抗流感药物及疫苗的研发上的作用,进行系统分析和综述。展开更多
目的阐明重组表达乙脑病毒非结构蛋白1(Non-structural protein 1,NS1)与包括乙脑病毒在内的多种蚊传黄病毒的抗原抗体反应,以及对乙脑病毒感染患者急性期血清标本的抗原抗体反应。方法本研究使用大肠杆菌原核表达载体(prokaryotic expr...目的阐明重组表达乙脑病毒非结构蛋白1(Non-structural protein 1,NS1)与包括乙脑病毒在内的多种蚊传黄病毒的抗原抗体反应,以及对乙脑病毒感染患者急性期血清标本的抗原抗体反应。方法本研究使用大肠杆菌原核表达载体(prokaryotic expression vector,pET)系统,重组表达乙脑病毒NS1基因。使用Western Blot实验方法检测重组表达蛋白质与多种,包括乙脑病毒在内的蚊传黄病毒抗体的反应,以及乙脑病毒感染患者急性期血清的抗原抗体反应。结果乙脑病毒(P3株)NS1基因表达产物以包涵体形式存在,经过变性和复性的表达产物在聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE,简称变性胶)显示为单一条带,分子量约为45000。进一步的抗原抗体分析结果表明,表达产物与乙脑病毒(蚊虫分离株,脑炎患者分离株)多克隆或者单克隆抗体以及乙脑病毒感染病人急性期血清标本可以获得完全一致的抗原/抗体杂交反应。重组表达产物与蚊传黄病毒,如登革病毒和黄热病毒多克隆抗体之间抗原/抗体杂交反应为阴性,但与西尼罗病毒和墨累谷脑炎病毒多克隆抗体呈现抗原/抗体阳性杂交反应。结论本文成功获得乙脑病毒NS1蛋白的重组表达并分析了重组蛋白对多种蚊传黄病毒及乙脑病毒感染患者标本之间的抗原/抗体反应,研究结果对于阐明乙脑病毒NS1蛋白与蚊传黄病毒之间抗原抗体反应提供了重要的基础数据。本研究所获得重组表达蛋白为进一步研究乙脑病毒NS1蛋白质功能等提供了重要的物质基础。展开更多
Zika virus(ZIKV)evolves non-structural proteins to evade immune response and ensure efficient replication in the host cells.Cholesterol metabolic enzyme 7-dehydrocholesterol reductase(DHCR7)was recently reported to im...Zika virus(ZIKV)evolves non-structural proteins to evade immune response and ensure efficient replication in the host cells.Cholesterol metabolic enzyme 7-dehydrocholesterol reductase(DHCR7)was recently reported to impact innate immune responses in ZIKV infection.However,the vital non-structural protein and mechanisms involved in DHCR7-mediated viral evasion are not well elucidated.In this study,we demonstrated that ZIKV infection facilitated DHCR7 expression.Notably,the upregulated DHCR7 in turn facilitated ZIKV infection and blocking DHCR7 suppressed ZIKV infection.Mechanically,ZIKV non-structural protein 4B(NS4B)interacted with DHCR7 to induce DHCR7 expression.Moreover,DHCR7 inhibited TANK-binding kinase 1(TBK1)and interferon regulatory factor 3(IRF3)phosphorylation,which resulted in the reduction of interferon-beta(IFN-β)and interferon-stimulated genes(ISGs)productions.Therefore,we propose that ZIKV NS4B binds to DHCR7 to repress TBK1 and IRF3 activation,which in turn inhibits IFN-βand ISGs,and thereby facilitating ZIKV evasion.This study broadens the insights on how viral non-structural proteins antagonize innate immunity to facilitate viral infection via cholesterol metabolic enzymes and intermediates.展开更多
目的获得流行性乙型脑炎(乙脑)病毒非结构蛋白1(Non-structural Protein 1,NS1)基因并在昆虫细胞中表达,为研制乙脑病毒基因工程疫苗奠定基础。方法逆转录-聚合酶链反应扩增乙脑病毒NS1基因并测序列,定向克隆入杆状病毒(Baculovirus,BV...目的获得流行性乙型脑炎(乙脑)病毒非结构蛋白1(Non-structural Protein 1,NS1)基因并在昆虫细胞中表达,为研制乙脑病毒基因工程疫苗奠定基础。方法逆转录-聚合酶链反应扩增乙脑病毒NS1基因并测序列,定向克隆入杆状病毒(Baculovirus,BV)转移载体pFastBac HTa,获得重组转移载体pFastBac-NS1。并将pFastBac-NS1转化到DH10Bac感受态细胞中,筛选到重组转座子rBacmid-NS1。在脂质体介导下将rBacmid-NS1转染粉蚊夜蛾(Trichoplusia ni,TN)细胞获得重组BV(rBV-NS1)。rBV-NS1感染TN细胞后,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulfate-Polyacryamide Gel Eleetrophoresis,SDS-PAG)、间接免疫荧光试验(Indirect Immunofluorescence Assay,IFA)和蛋白质印迹法(Western blotting,WB)检测NS1重组蛋白。结果成功克隆乙脑病毒NS1基因,SDS-PAGE、IFA和WB检测表明,NS1基因在昆虫细胞得到表达,能与乙脑患者血清发生特异性免疫反应,具有免疫原性。结论乙脑病毒NS1在昆虫细胞中表达,为研究NS1的生物学特性和研制基因工程疫苗奠定了基础。展开更多
Objective: To study protein-protein interaction between heterogeneous nuclear ribonucleoprotein H(hn RNP H) and Dengue virus(DENV) proteins. Methods: DENV proteins were screened against the host hn RNP H protein, in o...Objective: To study protein-protein interaction between heterogeneous nuclear ribonucleoprotein H(hn RNP H) and Dengue virus(DENV) proteins. Methods: DENV proteins were screened against the host hn RNP H protein, in order to identify the host-viral protein-protein interactions in DENV infected THP-1 cells by co-immunoprecipitation. The co-localization of the interacting proteins was further confirmed by immunofluorescence microscopy. Results: The host protein hn RNP H was found to interact with DENV nonstructural 1 protein and help the virus to multiply in the cell. Conclusions: The non-structural 1 glycoprotein is a key modulator of host immune response and is also involved in viral replication. Therefore, disruption of this key interaction between hn RNP H and DENV nonstructural 1 could be an important therapeutic strategy for management of DENV infection.展开更多
Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including tra...Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.展开更多
文摘甲型流感病毒(Influenza A virus,IAV)是引起每年季节性流感及偶尔的流感大流行的主要病原体,严重危害公共卫生和社会经济。IAV属于分节段的单股负链RNA病毒,其基因组编码多种不同的蛋白质,其中非结构蛋白1(non-structural protein 1,NS1)是IAV的调节宿主免疫应答和病毒致病性的重要毒力因子。NS1具有多种功能,它可以与多种宿主RNA及细胞因子相互作用,在增强病毒mRNA翻译、抑制I型干扰素和细胞凋亡等方面发挥重要作用,因此,深入研究IAV NS1在宿主细胞中的作用,对于更好地理解病毒感染和复制的机制是至关重要的。论文主要根据NS1的免疫逃逸机制、促进病毒复制和适应性进化,以及NS1作为新型治疗靶点在抗流感药物及疫苗的研发上的作用,进行系统分析和综述。
文摘目的阐明重组表达乙脑病毒非结构蛋白1(Non-structural protein 1,NS1)与包括乙脑病毒在内的多种蚊传黄病毒的抗原抗体反应,以及对乙脑病毒感染患者急性期血清标本的抗原抗体反应。方法本研究使用大肠杆菌原核表达载体(prokaryotic expression vector,pET)系统,重组表达乙脑病毒NS1基因。使用Western Blot实验方法检测重组表达蛋白质与多种,包括乙脑病毒在内的蚊传黄病毒抗体的反应,以及乙脑病毒感染患者急性期血清的抗原抗体反应。结果乙脑病毒(P3株)NS1基因表达产物以包涵体形式存在,经过变性和复性的表达产物在聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE,简称变性胶)显示为单一条带,分子量约为45000。进一步的抗原抗体分析结果表明,表达产物与乙脑病毒(蚊虫分离株,脑炎患者分离株)多克隆或者单克隆抗体以及乙脑病毒感染病人急性期血清标本可以获得完全一致的抗原/抗体杂交反应。重组表达产物与蚊传黄病毒,如登革病毒和黄热病毒多克隆抗体之间抗原/抗体杂交反应为阴性,但与西尼罗病毒和墨累谷脑炎病毒多克隆抗体呈现抗原/抗体阳性杂交反应。结论本文成功获得乙脑病毒NS1蛋白的重组表达并分析了重组蛋白对多种蚊传黄病毒及乙脑病毒感染患者标本之间的抗原/抗体反应,研究结果对于阐明乙脑病毒NS1蛋白与蚊传黄病毒之间抗原抗体反应提供了重要的基础数据。本研究所获得重组表达蛋白为进一步研究乙脑病毒NS1蛋白质功能等提供了重要的物质基础。
基金supported by the National Natural Science Foundation of China(81730061,81802008)the Guangdong Basic and Applied Basic Research Foundation(2021A1515011272).
文摘Zika virus(ZIKV)evolves non-structural proteins to evade immune response and ensure efficient replication in the host cells.Cholesterol metabolic enzyme 7-dehydrocholesterol reductase(DHCR7)was recently reported to impact innate immune responses in ZIKV infection.However,the vital non-structural protein and mechanisms involved in DHCR7-mediated viral evasion are not well elucidated.In this study,we demonstrated that ZIKV infection facilitated DHCR7 expression.Notably,the upregulated DHCR7 in turn facilitated ZIKV infection and blocking DHCR7 suppressed ZIKV infection.Mechanically,ZIKV non-structural protein 4B(NS4B)interacted with DHCR7 to induce DHCR7 expression.Moreover,DHCR7 inhibited TANK-binding kinase 1(TBK1)and interferon regulatory factor 3(IRF3)phosphorylation,which resulted in the reduction of interferon-beta(IFN-β)and interferon-stimulated genes(ISGs)productions.Therefore,we propose that ZIKV NS4B binds to DHCR7 to repress TBK1 and IRF3 activation,which in turn inhibits IFN-βand ISGs,and thereby facilitating ZIKV evasion.This study broadens the insights on how viral non-structural proteins antagonize innate immunity to facilitate viral infection via cholesterol metabolic enzymes and intermediates.
基金supported by the Defence Institute of Physiology and Allied SciencesDefence Research and Development Organization+1 种基金Ministry of DefenceIndia in the form of TASK-177
文摘Objective: To study protein-protein interaction between heterogeneous nuclear ribonucleoprotein H(hn RNP H) and Dengue virus(DENV) proteins. Methods: DENV proteins were screened against the host hn RNP H protein, in order to identify the host-viral protein-protein interactions in DENV infected THP-1 cells by co-immunoprecipitation. The co-localization of the interacting proteins was further confirmed by immunofluorescence microscopy. Results: The host protein hn RNP H was found to interact with DENV nonstructural 1 protein and help the virus to multiply in the cell. Conclusions: The non-structural 1 glycoprotein is a key modulator of host immune response and is also involved in viral replication. Therefore, disruption of this key interaction between hn RNP H and DENV nonstructural 1 could be an important therapeutic strategy for management of DENV infection.
文摘Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.
