外泌体中新城疫病毒F、W及V蛋白对病毒增殖的影响
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摘要
以外泌体跨膜蛋白CD63为靶点,利用protein A/G磁珠吸附抗体Fc端的特点,联合禽CD63抗体,建立了禽源外泌体分离方法。结果显示该方法可有效分离不含NDV粒子的外泌体(NDV Ex)。进一步检测发现,NDV Ex中含有NDV F、W和V蛋白。这些NDV Ex中包含的病毒蛋白能够通过降低干扰素的mRNA水平促进NDV复制,补充了外泌体在NDV感染中发挥作用的知识,为阐明新城疫病毒致病机制奠定了基础。
Exosomes are closely related to Newcastle disease virus(NDV) infection.However,there is no specific research on NDV-related exosomes because of the difficulty in separating virus particles from exosomes.To solve this problem,theexosome transmembrane protein CD63 was used as a target in our research.The proteinA/G magnetic beads were combined with anti-chicken CD63 antibody by catching the Fc end of the antibodies.The results show that this method can effectively separate exosomes(NDV Ex) which does not include NDV particles.Further testing revealed that NDV Ex contained NDV F,W and V proteins,which can promote NDV replication by reducing interferon mRNA levels.These results proved the importance of exosomes in NDV infection and provided a basis for elucidating the pathogenesis of NDV.
Exosomes are closely related to Newcastle disease virus(NDV) infection.However,there is no specific research on NDV-related exosomes because of the difficulty in separating virus particles from exosomes.To solve this problem,theexosome transmembrane protein CD63 was used as a target in our research.The proteinA/G magnetic beads were combined with anti-chicken CD63 antibody by catching the Fc end of the antibodies.The results show that this method can effectively separate exosomes(NDV Ex) which does not include NDV particles.Further testing revealed that NDV Ex contained NDV F,W and V proteins,which can promote NDV replication by reducing interferon mRNA levels.These results proved the importance of exosomes in NDV infection and provided a basis for elucidating the pathogenesis of NDV.
引文
[1] 邓紫艳,项勋,段纲,等.新城疫病毒分子生物学研究进展[J].上海畜牧兽医通讯,2017(1):11-13.
[2] 于洋,封振,何孔旺,等.新城疫病毒毒力分子机制研究进展[J].江苏农业学报,2013,29(2):435-439.
[3] 刘玉良,刘秀梵.新城疫病毒P基因的RNA编辑及其抗干扰素作用[J].动物医学进展,2004,25(6):1-3.
[4] 丁铲.新城疫病毒核衣壳蛋白的结构和功能[J].中国预防兽医学报,2011(1):80-84.
[5] ALEXANDER D J.Newcastle disease and other avian paramyxoviruses[J].Rev Sci Tech,2000,19(2):443-462.
[6] 刘晶雪,王志玉.新城疫病毒HN蛋白促细胞融合机制研究进展[J].病毒学报,2017(6):959-964.
[7] 沈咏舟,郑新勇.新城疫病毒F和HN蛋白的氨基酸序列对其毒力的影响[J].生命的化学,2000(5):220-222.
[8] 刘晶雪,王志玉.新城疫病毒HN蛋白促细胞融合机制研究进展[J].病毒学报,2017(6):959-964.
[9] 段云兵,汪军卿,仇旭升,等.新城疫病毒M蛋白通过自身核定位信号介导进入宿主细胞核[J].南京农业大学学报,2014,37(2):105-110.
[10] 段志强,邓珊珊,袁超,等.新城疫病毒M蛋白核定位信号突变降低病毒的致病性[J].畜牧兽医学报,2018,49(12):161-168.
[11] AUDSLEY M D,MOSELEY G W.Paramyxovirus evasion of innate immunity:Diverse strategies for common targets[J].World J Virol,2013,2(2):57.
[12] HUANG Z,KRISHNAMURTHY S,PANDA A,et al.Newcastle disease virus v protein is associated with viral pathogenesis and functions as an alpha interferon antagonist[J].J Virol,2003,77(16):8676-8685.
[13] 卢婉,杨人强,王伶.外泌体的研究进展[J].生命的化学,2013,51(4):438-442.
[14] 赵越,王超,陈和忠.外泌体生成和分泌机制的研究进展[J].解放军医学杂志,2017(12):1106-1109.
[15] FRüHBEIS C,FR?HLICH D,KUO W P,et al.Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte–neuron communication[J].PLoS Biol,2013,11(7):e1001604.
[16] REGEV-RUDZKI N,WILSON DANNY W,CARVALHO TERESA G,et al.Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles[J].Cell,2013,153(5):1120-33.
[17] WANG T,FANG L,ZHAO F,et al.Exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus (PRRSV)[J].J Virol,2017,92(4):JVI.01734-17.
[18] COSSET F L,DREUX M.HCV transmission by hepatic exosomes establishes a productive infection[J].J Hepatoly,2014,60(3):674-675.
[19] YANG Y,HAN Q,HOU Z,et al.Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction[J].Cell Mol Immunoly,2017,14(5):465-475.
[20] LENASSI M,CAGNEY G,LIAO M,et al.HIV NEF is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells[J].Traffic,2010,11(1):110-122.
[21] PIN L,MELISA K,HAN L S,et al.Progress in exosome isolation techniques[J].Theranostics,2017,7(3):789-804.
[22] 龚春梅,徐远飞,周继昌.外泌体分离与鉴定方法的研究进展[J].生命科学,2018,30(3):319-326.
[23] GREENING D W,XU R,JI H,et al.A Protocol for exosome isolation and characterization:evaluation of ultracentrifugation,density-gradient separation,and immunoaffinity capture methods[J].Methods Mol Biol,2015,1295:179-209.
[24] MA Z,RAMAKRISHNA S.Electrospun regenerated cellulose nanofiber affinity membrane functionalized with protein A/G for IgG purification[J].J Membrane Sci,2008,319(1/2):23-28.
[25] 江爱民,朱耐伟,朱勇喆.外泌体在病毒感染中的研究进展[J].生命的化学,2017(4):137-142.
[26] CHAHAR H S,CORSELLO T,KUDLICKI A S,et al.Respiratory syncytial virus infection changes cargo composition of exosome released from airway epithelial cells[J].Sci Rep,2018,8(1):387.
[27] 王露,刘卓然.EB病毒感染相关外泌体的研究进展[J].微生物学免疫学进展,2018,46(4):69-74.
[28] PLEET M L,ALLISON M,CATHERINE D M,et al.Ebola VP40 in Exosomes can cause immune cell dysfunction[J].Front Microbiol,2016(7):1765.
[29] 刘温泉,王远萍,邹年莉,等.新城疫病毒感染鸡胚成纤维细胞中免疫相关细胞因子表达的检测[J].中国家禽,2009,31(19):12-15.
[2] 于洋,封振,何孔旺,等.新城疫病毒毒力分子机制研究进展[J].江苏农业学报,2013,29(2):435-439.
[3] 刘玉良,刘秀梵.新城疫病毒P基因的RNA编辑及其抗干扰素作用[J].动物医学进展,2004,25(6):1-3.
[4] 丁铲.新城疫病毒核衣壳蛋白的结构和功能[J].中国预防兽医学报,2011(1):80-84.
[5] ALEXANDER D J.Newcastle disease and other avian paramyxoviruses[J].Rev Sci Tech,2000,19(2):443-462.
[6] 刘晶雪,王志玉.新城疫病毒HN蛋白促细胞融合机制研究进展[J].病毒学报,2017(6):959-964.
[7] 沈咏舟,郑新勇.新城疫病毒F和HN蛋白的氨基酸序列对其毒力的影响[J].生命的化学,2000(5):220-222.
[8] 刘晶雪,王志玉.新城疫病毒HN蛋白促细胞融合机制研究进展[J].病毒学报,2017(6):959-964.
[9] 段云兵,汪军卿,仇旭升,等.新城疫病毒M蛋白通过自身核定位信号介导进入宿主细胞核[J].南京农业大学学报,2014,37(2):105-110.
[10] 段志强,邓珊珊,袁超,等.新城疫病毒M蛋白核定位信号突变降低病毒的致病性[J].畜牧兽医学报,2018,49(12):161-168.
[11] AUDSLEY M D,MOSELEY G W.Paramyxovirus evasion of innate immunity:Diverse strategies for common targets[J].World J Virol,2013,2(2):57.
[12] HUANG Z,KRISHNAMURTHY S,PANDA A,et al.Newcastle disease virus v protein is associated with viral pathogenesis and functions as an alpha interferon antagonist[J].J Virol,2003,77(16):8676-8685.
[13] 卢婉,杨人强,王伶.外泌体的研究进展[J].生命的化学,2013,51(4):438-442.
[14] 赵越,王超,陈和忠.外泌体生成和分泌机制的研究进展[J].解放军医学杂志,2017(12):1106-1109.
[15] FRüHBEIS C,FR?HLICH D,KUO W P,et al.Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte–neuron communication[J].PLoS Biol,2013,11(7):e1001604.
[16] REGEV-RUDZKI N,WILSON DANNY W,CARVALHO TERESA G,et al.Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles[J].Cell,2013,153(5):1120-33.
[17] WANG T,FANG L,ZHAO F,et al.Exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus (PRRSV)[J].J Virol,2017,92(4):JVI.01734-17.
[18] COSSET F L,DREUX M.HCV transmission by hepatic exosomes establishes a productive infection[J].J Hepatoly,2014,60(3):674-675.
[19] YANG Y,HAN Q,HOU Z,et al.Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction[J].Cell Mol Immunoly,2017,14(5):465-475.
[20] LENASSI M,CAGNEY G,LIAO M,et al.HIV NEF is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells[J].Traffic,2010,11(1):110-122.
[21] PIN L,MELISA K,HAN L S,et al.Progress in exosome isolation techniques[J].Theranostics,2017,7(3):789-804.
[22] 龚春梅,徐远飞,周继昌.外泌体分离与鉴定方法的研究进展[J].生命科学,2018,30(3):319-326.
[23] GREENING D W,XU R,JI H,et al.A Protocol for exosome isolation and characterization:evaluation of ultracentrifugation,density-gradient separation,and immunoaffinity capture methods[J].Methods Mol Biol,2015,1295:179-209.
[24] MA Z,RAMAKRISHNA S.Electrospun regenerated cellulose nanofiber affinity membrane functionalized with protein A/G for IgG purification[J].J Membrane Sci,2008,319(1/2):23-28.
[25] 江爱民,朱耐伟,朱勇喆.外泌体在病毒感染中的研究进展[J].生命的化学,2017(4):137-142.
[26] CHAHAR H S,CORSELLO T,KUDLICKI A S,et al.Respiratory syncytial virus infection changes cargo composition of exosome released from airway epithelial cells[J].Sci Rep,2018,8(1):387.
[27] 王露,刘卓然.EB病毒感染相关外泌体的研究进展[J].微生物学免疫学进展,2018,46(4):69-74.
[28] PLEET M L,ALLISON M,CATHERINE D M,et al.Ebola VP40 in Exosomes can cause immune cell dysfunction[J].Front Microbiol,2016(7):1765.
[29] 刘温泉,王远萍,邹年莉,等.新城疫病毒感染鸡胚成纤维细胞中免疫相关细胞因子表达的检测[J].中国家禽,2009,31(19):12-15.