Name: production of high-titer infectious influenza pseudotyped particles with envelope glycoproteins from highly pathogenic h5n1 and avian h7n9 viruses
Uploaded: 2020-01-15T12:00:00
Description: Watch this Scientific Journal Video about Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses at JoVE.com

这项工作得到了浙江省医药卫生科技计划（授权号，2017KY538）、杭州市医药卫生科技计划（授权号，OO20190070）、杭州医药科学与技术重点项目（授权号，2014Z11）和杭州市社会发展与科学研究自主应用项目（授权号，20191203B134）。

1. 第1天：细胞培养和播种
<ol><li>在 60 mm 培养的 60 mm 培养皿中培养人类胚胎肾 （HEK） 293T/17 细胞，使用 Dulbeco 的改性必需介质 （DMEM） 辅以 10% 胎儿牛血清 （FBS） 和 100 U/mL 青霉素-链霉素 （DMEM 完整介质， DCM） 在 37°C， 5% 二氧化碳 （CO2） 培养箱中，直到约 80% 的汇合。 注：建议使用HEK 293T/17低通道单元。</li>
	<li>用5 mL的磷酸盐缓冲盐水（PBS）1x小心清洗细胞。 注：HEK 293T/17 单元?...

<ol>
	<li>Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology (6th). , (2013).</li><li>White, J. M., Delos, S. E., Brecher, M., Schornberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+and+mechanisms+of+viral+membrane+fusion+proteins:+multiple+variations+on+a+common+theme.">Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme.</a> Critical Reviews in Biochemistry and Molecular Biology. 43 (3), 189-219 (2008).</li><li>Bright, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-clade+protective+immune+responses+to+influenza+viruses+with+H5N1+HA+and+NA+elicited+by+an+influenza+virus-like+particle.">Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle.</a> PLoS One. 3 (1), 1501 (2008).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+pseudotyped+influenza+viral+particles+in+neutralizing+antibody+detection.">Reliability of pseudotyped influenza viral particles in neutralizing antibody detection.</a> PLoS One. 9 (12), 113629 (2014).</li><li>Wyatt, R., Sodroski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HIV-1+envelope+glycoproteins:+fusogens,+antigens,+and+immunogens.">The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens.</a> Science. 280 (5371), 1884-1888 (1998).</li><li>Joe, A. K., Foo, H. H., Kleeman, L., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transmembrane+domains+of+Sindbis+virus+envelope+glycoproteins+induce+cell+death.">The transmembrane domains of Sindbis virus envelope glycoproteins induce cell death.</a> Journal of Virology. 72 (5), 3935-3943 (1998).</li><li>Albecka, A., Laine, R. F., Janssen, A. F., Kaminski, C. F., Crump, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSV-1+Glycoproteins+Are+Delivered+to+Virus+Assembly+Sites+Through+Dynamin-Dependent+Endocytosis.">HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.</a> Traffic. 17 (1), 21-39 (2016).</li><li>Huang, A. S., Palma, E. L., Hewlett, N., Roizman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudotype+formation+between+enveloped+RNA+and+DNA+viruses.">Pseudotype formation between enveloped RNA and DNA viruses.</a> Nature. 252 (5485), 743-745 (1974).</li><li>Rubin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Control+of+Cellular+Susceptibility+to+Pseudotypes+of+Rous+Sarcoma+Virus.">Genetic Control of Cellular Susceptibility to Pseudotypes of Rous Sarcoma Virus.</a> Virology. 26, 270-276 (1965).</li><li>Steffen, I., Simmons, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudotyping+Viral+Vectors+With+Emerging+Virus+Envelope+Proteins.">Pseudotyping Viral Vectors With Emerging Virus Envelope Proteins.</a> Current Gene Therapy. 16 (1), 47-55 (2016).</li><li>Bartosch, B., Dubuisson, J., Cosset, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+hepatitis+C+virus+pseudo-particles+containing+functional+E1-E2+envelope+protein+complexes.">Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes.</a> Journal of Experimental Medicine. 197 (5), 633-642 (2003).</li><li>Bian, T., Zhou, Y., Bi, S., Tan, W., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCV+envelope+protein+function+is+dependent+on+the+peptides+preceding+the+glycoproteins.">HCV envelope protein function is dependent on the peptides preceding the glycoproteins.</a> Biochemical and Biophysical Research Communications. 378 (1), 118-122 (2009).</li><li>Gudima, S., Meier, A., Dunbrack, R., Taylor, J., Bruss, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+potentially+important+elements+of+the+hepatitis+B+virus+large+envelope+protein+are+dispensable+for+the+infectivity+of+hepatitis+delta+virus.">Two potentially important elements of the hepatitis B virus large envelope protein are dispensable for the infectivity of hepatitis delta virus.</a> Journal of Virology. 81 (8), 4343-4347 (2007).</li><li>Yoshida, Y., Emi, N., Hamada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSV-G-pseudotyped+retroviral+packaging+through+adenovirus-mediated+inducible+gene+expression.">VSV-G-pseudotyped retroviral packaging through adenovirus-mediated inducible gene expression.</a> Biochemical and Biophysical Research Communications. 232 (2), 379-382 (1997).</li><li>Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., Yee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+G+glycoprotein+pseudotyped+retroviral+vectors:+concentration+to+very+high+titer+and+efficient+gene+transfer+into+mammalian+and+nonmammalian+cells.">Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (17), 8033-8037 (1993).</li><li>Zhang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pseudoparticles+paired+with+hemagglutinin+and+neuraminidase+from+highly+pathogenic+H5N1+influenza+and+avian+influenza+A+(H7N9)+viruses.">Characterization of pseudoparticles paired with hemagglutinin and neuraminidase from highly pathogenic H5N1 influenza and avian influenza A (H7N9) viruses.</a> Virus Research. 253, 20-27 (2018).</li><li>Zhang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectivity+of+Pseudotyped+Particles+Pairing+Hemagglutinin+of+Highly+Pathogenic+Avian+Influenza+a+H5N1+Virus+with+Neuraminidases+of+The+2009+Pandemic+H1N1+and+a+Seasonal+H3N2.">Infectivity of Pseudotyped Particles Pairing Hemagglutinin of Highly Pathogenic Avian Influenza a H5N1 Virus with Neuraminidases of The 2009 Pandemic H1N1 and a Seasonal H3N2.</a> Journal of Bioterrorism &amp; Biodefense. 2, 104 (2011).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neuraminidases+from+the+highly+pathogenic+avian+H5N1+and+2009+pandemic+H1N1+influenza+A+viruses.">Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses.</a> PLoS One. 5 (12), 15825 (2010).</li><li>Nefkens, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinin+pseudotyped+lentiviral+particles:+characterization+of+a+new+method+for+avian+H5N1+influenza+sero-diagnosis.">Hemagglutinin pseudotyped lentiviral particles: characterization of a new method for avian H5N1 influenza sero-diagnosis.</a> Journal of Clinical Virology. 39 (1), 27-33 (2007).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinin+and+neuraminidase+matching+patterns+of+two+influenza+A+virus+strains+related+to+the+1918+and+2009+global+pandemics.">Hemagglutinin and neuraminidase matching patterns of two influenza A virus strains related to the 1918 and 2009 global pandemics.</a> Biochemical and Biophysical Research Communications. 387 (2), 405-408 (2009).</li><li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oseltamivir+boosts+2009+H1N1+virus+infectivity+in+vitro.">Oseltamivir boosts 2009 H1N1 virus infectivity in vitro.</a> Biochemical and Biophysical Research Communications. 390 (4), 1305-1308 (2009).</li><li>McKay, T., Patel, M., Pickles, R. J., Johnson, L. G., Olsen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+M2+envelope+protein+augments+avian+influenza+hemagglutinin+pseudotyping+of+lentiviral+vectors.">Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors.</a> Gene Therapy. 13 (8), 715-724 (2006).</li><li>Pan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+mediates+avian+influenza+H5N1+pseudotyped+particle-induced+lung+inflammation+through+NF-kappaB+and+p38+MAPK+signaling+pathways.">Autophagy mediates avian influenza H5N1 pseudotyped particle-induced lung inflammation through NF-kappaB and p38 MAPK signaling pathways.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 306 (2), 183-195 (2014).</li><li>Szecsi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+neutralising+antibodies+by+virus-like+particles+harbouring+surface+proteins+from+highly+pathogenic+H5N1+and+H7N1+influenza+viruses.">Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses.</a> Virology Journal. 3, 70 (2006).</li><li>Garcia, J. M., Lagarde, N., Ma, E. S., de Jong, M. D., Peiris, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+evaluation+of+an+influenza+A+(H5)+pseudotyped+lentiviral+particle-based+serological+assay.">Optimization and evaluation of an influenza A (H5) pseudotyped lentiviral particle-based serological assay.</a> Journal of Clinical Virology. 47 (1), 29-33 (2010).</li><li>Garcia, J. M., Lai, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+influenza+pseudotyped+lentiviral+particles+and+their+use+in+influenza+research+and+diagnosis:+an+update.">Production of influenza pseudotyped lentiviral particles and their use in influenza research and diagnosis: an update.</a> Expert Review of Anti-infective Therapy. 9 (4), 443-455 (2011).</li><li>Haynes, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza-pseudotyped+Gag+virus-like+particle+vaccines+provide+broad+protection+against+highly+pathogenic+avian+influenza+challenge.">Influenza-pseudotyped Gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge.</a> Vaccine. 27 (4), 530-541 (2009).</li><li>Schmeisser, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+mammalian+virus-like+particles+from+modified+vaccinia+virus+Ankara+vectors+expressing+influenza+H5N1+hemagglutinin+and+neuraminidase.">Production and characterization of mammalian virus-like particles from modified vaccinia virus Ankara vectors expressing influenza H5N1 hemagglutinin and neuraminidase.</a> Vaccine. 30 (23), 3413-3422 (2012).</li><li>Liu, Y. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+severe+acute+respiratory+syndrome+coronavirus+(SARS-CoV)+S+glycoprotein+and+influenza+matrix+1+efficiently+form+virus-like+particles+(VLPs)+that+protect+mice+against+challenge+with+SARS-CoV.">Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV.</a> Vaccine. 29 (38), 6606-6613 (2011).</li><li>Moeschler, S., Locher, S., Conzelmann, K. K., Kramer, B., Zimmer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Lyssavirus-Neutralizing+Antibodies+Using+Vesicular+Stomatitis+Virus+Pseudotype+Particles.">Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles.</a> Viruses. 8 (9), 254 (2016).</li><li>Lai, A. L., Millet, J. K., Daniel, S., Freed, J. H., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SARS-CoV+Fusion+Peptide+Forms+an+Extended+Bipartite+Fusion+Platform+that+Perturbs+Membrane+Order+in+a+Calcium-Dependent+Manner.">The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner.</a> Journal of Molecular Biology. 429 (24), 3875-3892 (2017).</li><li>Millet, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+East+respiratory+syndrome+coronavirus+infection+is+inhibited+by+griffithsin.">Middle East respiratory syndrome coronavirus infection is inhibited by griffithsin.</a> Antiviral Research. 133, 1-8 (2016).</li><li>Millet, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Pseudotyped+Particles+to+Study+Highly+Pathogenic+Coronaviruses+in+a+Biosafety+Level+2+Setting.">Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting.</a> Journal of Visualized Experiments. (145), e59010 (2019).</li><li>Ma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+leukemia+virus+pseudotypes+of+La+Crosse+and+Hantaan+Bunyaviruses:+a+system+for+analysis+of+cell+tropism.">Murine leukemia virus pseudotypes of La Crosse and Hantaan Bunyaviruses: a system for analysis of cell tropism.</a> Virus Research. 64 (1), 23-32 (1999).</li><li>Wool-Lewis, R. J., Bates, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Ebola+virus+entry+by+using+pseudotyped+viruses:+identification+of+receptor-deficient+cell+lines.">Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines.</a> Journal of Virology. 72 (4), 3155-3160 (1998).</li><li>Chen, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+design+of+more+effective+avian+replication-incompetent+retroviral+vectors.">Production and design of more effective avian replication-incompetent retroviral vectors.</a> Developmental Biology. 214 (2), 370-384 (1999).</li><li>Kaku, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+generation+of+pseudotype-based+serum+neutralization+assay+for+Nipah+virus+antibodies:+sensitive+and+high-throughput+analysis+utilizing+secreted+alkaline+phosphatase.">Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.</a> Journal of Virological Methods. 179 (1), 226-232 (2012).</li><li>Rudiger, D., Kupke, S. Y., Laske, T., Zmora, P., Reichl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+modeling+of+influenza+A+virus+replication+in+cell+cultures+predicts+infection+dynamics+for+highly+different+infection+conditions.">Multiscale modeling of influenza A virus replication in cell cultures predicts infection dynamics for highly different infection conditions.</a> PLOS Computational Biology. 15 (2), 1006819 (2019).</li><li>Petiot, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+viruses+production:+Evaluation+of+a+novel+avian+cell+line+DuckCelt(R)-T17.">Influenza viruses production: Evaluation of a novel avian cell line DuckCelt(R)-T17.</a> Vaccine. 36 (22), 3101-3111 (2018).</li></ol>

在此协议中，我们描述了在BSL-2设置下产生流感病毒伪型颗粒（pp）的方法。报告员质粒 pcDNA-GFP 被合并到 pps 中，可用于在感染性测定中通过 FACS 对 pps 进行量化。我们选择了两种类型的易感细胞系，因为它们在流感研究中被广泛使用。MDCK细胞将很好地控制这些研究中使用的可变永生的人类细胞。
该协议基于逆转录病毒MLV，它可以合并GFP报告器，并在细胞膜上产生芽。这项技...

病毒粒子的使命是将其基因组从受感染的宿主细胞输送到未受感染的宿主细胞，并将其以具有复制能力的形式1的形式输送到细胞质或细胞核中。这个过程最初由结合到宿主细胞受体触发，然后是病毒和细胞膜的融合。对于包络病毒，如流感病毒，尖峰糖蛋白负责受体结合和融合1，2。病毒包络糖蛋白（例如，热原、抗原）涉及许多重要特性和事件，如病毒生命周期启动（结合和融合）、病毒发病机制、免疫原性、宿主细胞凋亡和细胞代谢、细胞内分泌途径以及物种间传播和重组1、3、4、5、6、7。研究病毒包络糖蛋白将有助于我们了解病毒感染过程的许多方面。伪型病毒粒子（pp），也称为伪病毒或伪粒子，可以通过伪打字技术8，9，10生成。这项技术已用于开发多种病毒的伪型颗粒，包括丙型肝炎11、12、乙型肝炎13、脉体口腔炎病毒（VSV）14、15和流感病毒16、17、18、19。该技术基于慢病毒或其他逆转录病毒的Gag-Pol蛋白。
假型病毒颗粒可以通过将病毒包络糖蛋白表达质粒、缺少包络基因的抗逆转录病毒包装质粒和单独的报告质粒转化为pp生产者细胞，从而获得三质粒系统。逆转录病毒由Gag蛋白组装而成，它从表达病毒包络蛋白1的受感染细胞膜中发芽。因此，利用逆转录病毒Gag蛋白在表达流感HA和NA的细胞膜上产生芽是可能的。在我们以前的研究中，所有组合中的HAs/NA都是功能性的，能够在病毒生命周期16、17、18、20、21中执行相应的功能。这些pps用于研究流感生物特性，包括血凝、神经氨酸酶活性、HA受体结合对流和传染性。由于HA和NA都是病毒生命周期中重要的表面功能蛋白，从不同流感毒株衍生的不匹配的HA和NA可以部分地证明它们之间的重组。在这里，我们通过使用三个质粒伪型粒系统组合两个HA和两个NA（源自HPAI H5N1病毒株和H7N9染色）来生成八种类型的流感pps。这八种类型的pps包括两个原生pps，H5N1pp，H7N9pp;两个不匹配的 pps，（H5_N9）pp，（H7+N1）pp;和4pps只含有单糖蛋白（HA或NA），H5pp，N1pp，H7pp，N9pp。所有野生流感病毒株的研究都应在生物安全3级（BSL-3）实验室进行。伪型病毒颗粒技术可用于在生物安全级别 2 （BSL-2） 设置中包装人工病毒。因此，pps 是研究流感病毒过程的一个更安全和有用的工具，具体取决于其两种主要糖蛋白：血凝素 （HA） 和神经氨酸酶 （NA）。
该协议描述了这些pps的生成与三质粒共转染策略（在图1概述），如何量化pps和感染性检测。pp生产涉及三种质粒（图1）。编码逆转录病毒Gag-Pol蛋白的gag-pol基因从逆转录病毒包装试剂盒中克隆，并插入pcDNA 3.1质粒，并命名为pcDNA-Gag-Pol。编码绿色荧光蛋白的增强型绿色荧光蛋白（eGFP）基因从pTRE-EGFP载体克隆，插入pcDNA 3.1质粒，称为pcDNA-GFP。在克隆过程中，通过引信添加包装信号（*）序列。HA和NA基因被克隆成pVRC质粒，分别命名为pVRC-HA和pVRC-NA。最后一个质粒编码融合蛋白，可以替换为任何其他感兴趣的融合蛋白。我们的伪打字平台包括两个糖蛋白表达质粒：pVRC-HA和pVRC-NA。这可以简化在BSL-2设置中不同病毒株之间的重新分类研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Millipore</td>
			<td>70664</td>
			<td>Effective viscosity reduction and removal of nucleic acids from protein solutions</td>
		</tr>
		<tr>
			<td>Clear Flat Bottom Polystyrene TC-treated Microplates (96-well)</td>
			<td>Corning</td>
			<td>3599</td>
			<td>Treated for optimal cell attachment 
			Sterilized by gamma radiation and certified nonpyrogenic 
			Individual alphanumeric codes for well identification</td>
		</tr>
		<tr>
			<td>Clear TC-treated Multiple Well Plates (6-wells)</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Individual alphanumerical codes for well identification 
			Treated for optimal cell attachment 
			Sterilized by gamma irradiation</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified essential medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>A widely used basal medium for supporting the growth of many different mammalian cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Excell</td>
			<td>FND500</td>
			<td>fetal bovine sera that can offer excellent value for basic cell culture, specialty research, and specific assays</td>
		</tr>
		<tr>
			<td>Fluorescence Activated Cell Sorting (FACS)</td>
			<td>Beckman coulter</td>
			<td>cytoflex</td>
			<td></td>
		</tr>
		<tr>
			<td>Human alveolar adenocarcinoma A549 cells</td>
			<td>ATCC</td>
			<td>CRM-CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Human embryonic kidney (HEK) HEK-293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td>A versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines</td>
		</tr>
		<tr>
			<td>Inverted fluorescent biological microscope</td>
			<td>Olympus</td>
			<td>BX51-32P01-FLB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Olympus</td>
			<td>CKX31-12PHP</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td>Rapid, sensitive and precise probe-based qPCR detection and quantitation of target RNA targets.</td>
		</tr>
		<tr>
			<td>Luna Universal Probe One-Step RT-qPCR Kit</td>
			<td>NEB</td>
			<td>E3006L</td>
			<td>Will withstand up to 14,000 RCF 
			RNase-/DNase-free Nonpyrogenic</td>
		</tr>
		<tr>
			<td>Madin-Darby Canine Kidney (MDCK) cells</td>
			<td>ATCC</td>
			<td>CCL-34</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxyClear Snaplock Microcentrifuge Tube (1.5 mL)</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>33 mm, gamma sterilized</td>
		</tr>
		<tr>
			<td>Millex-HV Syringe Filter Unit, 0.45 &#181;m, PVDF</td>
			<td>Millipore</td>
			<td>SLHV033RS</td>
			<td>an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in cell growth rate or morphology. Opti-MEM I medium is also recommended for use with cationic lipid transfection reagents, such as Lipofectamine reagent.</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>11058021</td>
			<td>The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria.</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Maximum RCF is 12,500 xg 
			Temperature range from -80 &#176;C to 120 &#176;C 
			RNase-/DNase-free 
			Sterile</td>
		</tr>
		<tr>
			<td>PP Centrifuge Tubes (15 mL)</td>
			<td>Corning</td>
			<td>430791</td>
			<td>a stable and highly reactive serine protease</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Beyotime</td>
			<td>ST532</td>
			<td>Treated for optimal cell attachment 
			Sterilized by gamma radiation and certified nonpyrogenic</td>
		</tr>
		<tr>
			<td>TC-treated Culture Dish (60mm)</td>
			<td>Corning</td>
			<td>430166</td>
			<td>Trypsin from bovine pancreas 
			TPCK Treated, essentially salt-free, lyophilized powder, &#8805;10,000 BAEE units/mg protein</td>
		</tr>
		<tr>
			<td>TPCK-trypsin</td>
			<td>Sigma</td>
			<td>T1426</td>
			<td>This liquid formulation of trypsin contains EDTA and phenol red. Gibco Trypsin-EDTA is made from trypsin powder, an irradiated mixture of proteases derived from porcine pancreas. Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation. The trypsin concentration required for dissociation varies with cell type and experimental requirements.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of high-titer infectious influenza pseudotyped particles with envelope glycoproteins from highly pathogenic h5n1 and avian h7n9 viruses

H5N1型禽流感病毒（H5N1）和H7N9偶尔直接传染给人类及其杀伤力，是严重的公共卫生问题，表明有可能发生疫情。然而，我们对病毒的分子理解还很原始，有必要研究其包络蛋白作为治疗靶点的生物特性，并制定控制感染的策略。我们开发了一个固体病毒伪型颗粒（pp）平台来研究禽流感病毒，包括其血凝素（HA）和神经氨酸酶（NA）包络糖蛋白的功能分析，HA和NA的重组特性，受体，营养，中和抗体，诊断，感染，用于药物开发和疫苗设计。在这里，我们描述了一个实验程序，以建立从两种甲型流感菌株（HAPI H5N1和2013年禽流感H7N9）的包络糖蛋白（HA，NA）的pps。他们的一代是基于一些病毒的能力，如鼠白血病病毒（MLV），将包络糖蛋白纳入一个pp。此外，我们还详细介绍了这些 pps 如何用 RT-qPCR 进行量化，以及根据 HA 和 NA 的来源对原生病毒和不匹配病毒 pp 的感染性检测。该系统是高度灵活和适应性强，可用于建立病毒pps与包络糖蛋白，可以纳入任何其他类型的包络病毒。因此，该病毒粒子平台可用于研究野生病毒的许多研究。

根据上述一般程序，我们生成了 10 种类型的 pps，结合两组 HA/NA 或 VSV-G 糖蛋白或无包络糖蛋白（如表 1所示）。其中七种具有传染性。含有无包络糖蛋白或仅含有NA的pps在这里没有表现出任何感染力。图1概述了流感pp的生产工艺。pps的透射电子显微图（例如，H5N1pp）如图3所示。这些pps的传染性测定结果如图4所示。七?...

Watch this Scientific Journal Video about Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses at JoVE.com

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

该协议描述了一个实验过程，以产生高蒂特传染性病毒伪型颗粒（pp），包络糖蛋白从两个流感A毒株，以及如何确定其传染性。该协议具有很强的适应性，可开发具有不同包络糖蛋白的任何其他类型的包络病毒的pps。

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

这种产生流感病毒PP并确定其感染率的技术可以简化BSL-2城市流感病毒的研究。我们根据 PPS 的 qRT-PCR 数据对感染的 PPS 进行规范化。对两个糖蛋白表达质粒进行编码，可以简化病毒再感染的研究。细胞播种后一天，在倒置的光显微镜下检查细胞形态和密度。理想情况下，细胞在转染时应大约为85%的汇合。每井用一毫升无血清DMEM代替介质，然后将盘子放回培养箱中。对于每一个细胞的转染井，稀释8微升的转染试剂到150微升的体积与减少血清介质。轻轻混合溶液，让它在室温下坐五分钟。同时，将2.5微克质粒DNA稀释成158微升的血清介质。经过5分钟的孵育，将稀释的DNA与稀释的转染试剂混合，轻轻混合溶液，在室温下保持15分钟。将脱氧核糖核酸脂复合物加入到相应的孔中，与无血清介质中的细胞一起加入。然后来回摇晃，轻轻混合盘子。在37摄氏度和5%的二氧化碳下孵育4至6小时。孵育后，取出介质，每井用两毫升DMEM代替。然后再孵育36至48小时。每井1万个细胞和96个井板播种每种类型的易感细胞。然后在37摄氏度和5%的二氧化碳孵化器中过夜。在转染后的第三天，检查应浅粉色或微橙色的介质颜色，并在 440 至 460 纳米波长下用倒置荧光生物显微镜检查细胞。转染后38至48小时，通过0.45微米聚乙烯氟化物滤芯将pseuotype颗粒或PPS通过，将其清除细胞碎片，然后将其分成小体积的等分物，储存在零下80摄氏度。为了量化PPS，将30微升纯化病毒颗粒转移到1.5微升无RNase管中，并加入1微升的苯松酶核酸酶。在37摄氏度下孵育管子一小时，以消除任何DNA和RNA污染。孵育后，将样品冷冻至零下70摄氏度，以活性核酸酶，并加入两微升蛋白酶K.孵育混合物30分钟，消化包络蛋白并释放CMV-GFP RNA。然后在100摄氏度下使蛋白酶K处于非活性状态3分钟。使用文本手稿中指定的底图和探针中的通用探头一步 RT-qPCR 套件使用定量逆转录RTPCR量化PPS。将PPS归化为每毫升10至5个RNA拷贝的4倍。然后将TPCK-trypsin加入到含有H7N9下凝素的PPS中，最终浓度为每毫升10微克。在37摄氏度下孵育PPS一小时，形成功能亚单位HA1和HA2。将规范化的PPS与DMEM介质以一比一的比例混合，然后将含有易感细胞的板带到生物安全柜。吸上一液，用100微升预热PBS洗涤细胞一次。在每个井中加入100微升的PPS-DMEM混合物，将每种PPS的感染性测试三分到一个易感细胞系。在37摄氏度和5%的二氧化碳下孵育4至6小时。然后吸制上一液，并更换100微升DCM孵化板再孵化24至36小时，然后进行感染检测。我们的结果是PPS现在被认为是传染性的人。因此，建议采取必要的安全防护程序，如戴口罩和在生物安全柜中进行感染性检测。该协议产生10种类型的伪型颗粒或PPS与两组血凝素和神经氨酸囊性口腔炎病毒G糖蛋白或无包糖蛋白。用透射电子显微镜对PPS进行成像，并在两个细胞系，A549和MDCK中评价了其感染率。在H5的PPS组，H5N1、H5加N9和H5的感染率在细胞系A549和40%5%和5%的MDCK中分别约为90%18%和10%。在H7的PPS组，H7N9、H7加N1和H7的感染率在细胞系A549和8%4%和1%的MDCK中分别约为10%7%和1%。A549和MDCK细胞的感染性约为21%和16%。而三角洲包络糖蛋白PP没有表现出感染性。请记住，PP 生产商 HEK-293T/17 细胞构成此程序的基础。因此，最佳生长教我们 HEK-293T/17 细胞是最重要的。按照此过程或其他一些测试（如 L2）可接受边界测定。这种测定几乎可以消除受体偏好的生物特性，该模式将揭示PPS的tropism。自从他们从流感病毒中克隆出的糖蛋白被克隆成两个加最大值。HA和NA蛋白可以单独研究，因此我们在它们之间重新分类。突变体和成熟效应可以探索。

Research

JoVE Journal

Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

科研

免疫与感染

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

禽流感监测与FTA卡：实地方法，生物安全，和运输问题解决了

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

包装艾滋病毒或FIV的基于Lentivector表达结构与VSV-G假型病毒颗粒转导

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

造型埃博拉病毒的生命周期在生物安全二级条件下的病毒样颗粒包含Tetracistronic小基因组

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

治疗核黄素血小板制品和紫外线光：对有效性高滴度细菌污染

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...