家蚕IAP互作蛋白的筛选、鉴定及其对BmNPV增殖的影响
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摘要
【目的】细胞凋亡作为昆虫免疫应答的重要组成部分,在病毒与宿主相互作用的过程中扮演着重要角色,以细胞凋亡相关基因为研究对象对阐明其在病毒感染过程中的作用具有重要的参考价值。本研究旨在筛选家蚕凋亡抑制蛋白(Bombyx mori inhibitor of apoptosis protein,BmIAP)的相互作用蛋白,并验证其在家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)侵染过程中与Bmiap的相互调控关系及在BmNPV增殖过程中的作用,为探明宿主与BmNPV相互作用的分子机制提供理论依据。【方法】利用免疫共沉淀技术筛选在BmNPV侵染家蚕细胞过程中与BmIAP相互作用的蛋白,对获得的特异性差异条带进行LC-MS/MS蛋白质谱分析,根据蛋白分子量大小并利用生物信息学方法对获得的蛋白进行鉴定,确定候选基因;通过分子克隆技术对候选基因进行克隆,利用SMART在线工具及BioEdit分别对候选基因的结构域进行预测及多序列比对分析;通过免疫荧光试验验证BmIAP和候选蛋白的细胞共定位情况,并进一步利用免疫共沉淀验证它们的相互作用;分别通过真核表达和基于CRISPR/Cas9的基因编辑系统对候选基因进行过表达和敲除,利用qRT-PCR技术检测相应基因的表达情况,以确定Bmiap和Bmpp5在BmNPV侵染过程中的调控关系;同样,在过表达和敲除Bmpp5后检测杆状病毒Vp39的表达量,以确定Bmpp5对BmNPV增殖的影响。【结果】通过免疫共沉淀获得7个可能与BmIAP相互作用的宿主蛋白和1个BmNPV蛋白,进一步分析确定1个与凋亡相关的候选基因Bmpp5;Bmpp5的开放阅读框为1 473 bp,编码490个氨基酸,预测的蛋白分子量约为56 kD,含3个TRP结构域和1个PP2Ac结构域,在昆虫间具有较高的保守性;免疫荧光检测证明BmIAP和BmPP5共定位于细胞质中,免疫共沉淀结果表明两者可以相互作用;在BmNPV侵染过程中,过表达Bmiap后,Bmpp5的表达量显著上调,而敲除Bmiap后,Bmpp5的表达量显著下调,表明Bmiap能够促进Bmpp5的表达;同时,过表达Bmpp5后,Bmiap显著上调表达,而敲除Bmpp5后,Bmiap显著下调表达,表明Bmpp5同样能够促进Bmiap的表达;过表达Bmpp5能够促进BmNPV的增殖,敲除Bmpp5能够抑制BmNPV的增殖,表明Bmpp5的表达利于BmNPV的增殖。【结论】鉴定了1个能够与BmIAP相互作用的蛋白BmPP5,且该蛋白在昆虫中高度保守;在BmNPV侵染过程中,Bmiap和Bmpp5能够相互促进,且Bmpp5能够促进BmNPV的复制增殖。
【Objective】As an important part of insect immune response, apoptosis plays an important role in the interaction between virus and host. The study of apoptosis-related genes is of great value in elucidating their roles in the process of virus infection. The objective of this study is to screen the interaction proteins of Bombyx mori inhibitor of apoptosis proteins(BmIAP), verify the regulatory relationships between them and Bmiap in the process of B. mori nucleopolyhedrovirus(BmNPV) infection and their roles in the proliferation of BmNPV, and to provide a theoretical basis for exploring the molecular mechanism of the interaction between host and BmNPV.【Method】The proteins interacting with BmIAP in the process of BmNPV infection in B. mori cells were screened by immunoprecipitation, and the specific differential bands were analyzed by LC-MS/MS. The obtained proteins were identified according to the molecular weight of the protein and bioinformatics method. The candidate gene was obtained by molecular cloning technique, and the domain of the candidate gene was predicted by SMART online tools and the multiple sequence alignment analysis was performed with BioEdit. Co-localization of BmIAP and candidate protein was verified by immunofluorescence assay, and their interaction was further verified by immunoprecipitation. The candidate gene was overexpressed and knocked out by eukaryotic expression and CRISPR/Cas9 gene editing system, respectively. The expression of corresponding genes was detected by qRT-PCR to determine the regulatory relationship between Bmiap and Bmpp5 in the process of BmNPV infection. Similarly, the expression of baculovirus Vp39 was detected after overexpression and knockout of Bmpp5 to determine the effect of Bmpp5 on the proliferation of BmNPV.【Result】Seven host proteins and one BmNPV protein which may interact with BmIAP were obtained by immunoprecipitation and further analysis identified a candidate gene Bmpp5 associated with apoptosis. The open reading frame(ORD) of Bmpp5 is 1 473 bp, encoding 490 amino acids. The predicted molecular weight of BmPP5 is about 56 kD. It contains three TRP domains and one PP2 Ac domain. It is highly conserved among insects. Immunofluorescence assay showed that BmIAP and BmPP5 were co-localized in cytoplasm, and the results of immunoprecipitation showed that they could interact with each other. In the process of BmNPV infection, after overexpression of Bmiap, the expression of Bmpp5 was significantly up-regulated, while after knockout of Bmiap, the expression of Bmpp5 was significantly down-regulated, suggesting that Bmiap could promote the expression of Bmpp5. Bmiap was significantly up-regulated after overexpression of Bmpp5, while Bmiap was significantly down-regulated after knockout of Bmpp5, suggesting that Bmpp5 could also promote the expression of Bmiap. Overexpression of Bmpp5 could promote the proliferation of BmNPV, and knockdown of Bmpp5 could inhibit the proliferation of BmNPV, indicating that the expression of Bmpp5 was conducive to the proliferation of BmNPV. 【Conclusion】A protein BmPP5, which interacts with BmIAP was identified and highly conserved in insects. In the process of BmNPV infection, Bmiap and Bmpp5 can promote each other. It is also proved that Bmpp5 can promote the replication and proliferation of BmNPV.
【Objective】As an important part of insect immune response, apoptosis plays an important role in the interaction between virus and host. The study of apoptosis-related genes is of great value in elucidating their roles in the process of virus infection. The objective of this study is to screen the interaction proteins of Bombyx mori inhibitor of apoptosis proteins(BmIAP), verify the regulatory relationships between them and Bmiap in the process of B. mori nucleopolyhedrovirus(BmNPV) infection and their roles in the proliferation of BmNPV, and to provide a theoretical basis for exploring the molecular mechanism of the interaction between host and BmNPV.【Method】The proteins interacting with BmIAP in the process of BmNPV infection in B. mori cells were screened by immunoprecipitation, and the specific differential bands were analyzed by LC-MS/MS. The obtained proteins were identified according to the molecular weight of the protein and bioinformatics method. The candidate gene was obtained by molecular cloning technique, and the domain of the candidate gene was predicted by SMART online tools and the multiple sequence alignment analysis was performed with BioEdit. Co-localization of BmIAP and candidate protein was verified by immunofluorescence assay, and their interaction was further verified by immunoprecipitation. The candidate gene was overexpressed and knocked out by eukaryotic expression and CRISPR/Cas9 gene editing system, respectively. The expression of corresponding genes was detected by qRT-PCR to determine the regulatory relationship between Bmiap and Bmpp5 in the process of BmNPV infection. Similarly, the expression of baculovirus Vp39 was detected after overexpression and knockout of Bmpp5 to determine the effect of Bmpp5 on the proliferation of BmNPV.【Result】Seven host proteins and one BmNPV protein which may interact with BmIAP were obtained by immunoprecipitation and further analysis identified a candidate gene Bmpp5 associated with apoptosis. The open reading frame(ORD) of Bmpp5 is 1 473 bp, encoding 490 amino acids. The predicted molecular weight of BmPP5 is about 56 kD. It contains three TRP domains and one PP2 Ac domain. It is highly conserved among insects. Immunofluorescence assay showed that BmIAP and BmPP5 were co-localized in cytoplasm, and the results of immunoprecipitation showed that they could interact with each other. In the process of BmNPV infection, after overexpression of Bmiap, the expression of Bmpp5 was significantly up-regulated, while after knockout of Bmiap, the expression of Bmpp5 was significantly down-regulated, suggesting that Bmiap could promote the expression of Bmpp5. Bmiap was significantly up-regulated after overexpression of Bmpp5, while Bmiap was significantly down-regulated after knockout of Bmpp5, suggesting that Bmpp5 could also promote the expression of Bmiap. Overexpression of Bmpp5 could promote the proliferation of BmNPV, and knockdown of Bmpp5 could inhibit the proliferation of BmNPV, indicating that the expression of Bmpp5 was conducive to the proliferation of BmNPV. 【Conclusion】A protein BmPP5, which interacts with BmIAP was identified and highly conserved in insects. In the process of BmNPV infection, Bmiap and Bmpp5 can promote each other. It is also proved that Bmpp5 can promote the replication and proliferation of BmNPV.
引文
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[3]VANDERGAAST R,SCHULTZ K L,CERIO R J,FRIESEN P D.Active depletion of host cell inhibitor-of-apoptosis proteins triggers apoptosis upon baculovirus DNA replication.Journal of Virology,2011,85(16):8348-8358.
[4]MITCHELL J K,FRIESEN P D.Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication.Journal of Virology,2012,86(24):13542-13553.
[5]SALEM T Z,ZHANG F,XIE Y,THIEM S M.Comprehensive analysis of host gene expression in Autographa californica nucleopolyhedrovirus-infected Spodoptera frugiperda cells.Virology,2011,412(1):167-178.
[6]BAO Y Y,LV Z Y,LIU Z B,XUE J,XU Y P,ZHANG C X.Comparative analysis of Bombyx mori nucleopolyhedrovirus responsive genes in fat body and haemocyte of B.mori resistant and susceptible strains.Insect Molecular Biology,2010,19(3):347-358.
[7]MEHRABADI M,HUSSAIN M,ASGARI S.MicroRNAome of Spodoptera frugiperda cells(Sf9)and its alteration following baculovirus infection.The Journal of General Virology,2013,94(6):1385-1397.
[8]YU X,ZHOU Q,LI S C,LUO Q,CAI Y,LIN W C,CHEN H,YANGY,HU S,YU J.The silkworm(Bombyx mori)microRNAs and their expressions in multiple developmental stages.PLoS ONE,2008,3(8):e2997.
[9]XUE J,QIAO N,ZHANG W,CHENG R L,ZHANG X Q,BAO Y Y,XU Y P,GU L Z,HAN J D,ZHANG C X.Dynamic interactions between Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis.Journal of Virology,2012,86(13):7345-7359.
[10]蒋亚明,董战旗,陈婷婷,胡楠,董非凡,黄亮,唐良彤,潘敏慧.杆状病毒LEF-11蛋白自身相互作用关键区域的鉴定.中国农业科学,2017,50(20):4028-4035.JIANG Y M,DONG Z Q,CHEN T T,HU N,DONG F F,HUANG L,TANG L T,PAN M H.Identification the key areas of Bombyx mori nucleopolyhedrovirus LEF-11 self-interaction.Scientia Agricultura Sinica,2017,50(20):4028-4035.(in Chinese)
[11]CLEM R J.Baculoviruses and apoptosis:a diversity of genes and responses.Current Drug Targets,2007,8(10):1069-1074.
[12]MONTEIRO F,CARINHAS N,CARRONDO M J,BERNAL V,ALVES P M.Toward system-level understanding of baculovirus-host cell interactions:from molecular fundamental studies to large-scale proteomics approaches.Frontiers in Microbiology,2012,3:Article391.
[13]GUY M P,FRIESEN P D.Reactive-site cleavage residues confer target specificity to baculovirus P49,a dimeric member of the P35family of caspase inhibitors.Journal of Virology,2008,82(15):7504-7514.
[14]HUANG N,WU W,YANG K,PASSARELLI A L,ROHRMANN G F,CLEM R J.Baculovirus infection induces a DNA damage response that is required for efficient viral replication.Journal of Virology,2011,85(23):12547-12556.
[15]MITCHELL J K,BYERS N M,FRIESEN P D.Baculovirus F-box protein LEF-7 modifies the host DNA damage response to enhance virus multiplication.Journal of Virology,2013,87(23):12592-12599.
[16]SHIRATA N,IKEDA M,KOBAYASHI M.Identification of a Hyphantria cunea nucleopolyhedrovirus(NPV)gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPVmultiplication in a B.mori cell line.Virology,2010,398(2):149-157.
[17]KANNAN R P,HENSLEY L L,EVERS L E,LEMON S M,MCGIVERN D R.Hepatitis C virus infection causes cell cycle arrest at the level of initiation of mitosis.Journal of Virology,2011,85(16):7989-8001.
[18]DING L,HUANG Y,DAI M,ZHAO X,DU Q,DONG F,WANG L,HUO R,ZHANG W,XU X,TONG D.Transmissible gastroenteritis virus infection induces cell cycle arrest at S and G2/M phases via p53-dependent pathway.Virus Research,2013,178(2):241-251.
[19]EVERETT H,MCFADDEN G.Apoptosis:an innate immune response to virus infection.Trends in Microbiology,1999,7(4):160-165.
[20]HUANG Y,LIU H,LI S,TANG Y,WEI B,YU H,WANG C.MAVS-MKK7-JNK2 defines a novel apoptotic signaling pathway during viral infection.PLoS Pathogens,2014,10(3):e1004020.
[21]ZMASEK C M,GODZIK A.Evolution of the animal apoptosis network//BAEHRECKE E H,GREEN D R,KORNBLUTH S,SALVESEN G S.Cold Spring Harbor Perspectives in Biology,2013,5:a008649.
[22]ZHANG J Y,PAN M H,SUN Z Y,HUANG S J,YU Z S,LIU D,ZHAO D H,LU C.The genomic underpinnings of apoptosis in the silkworm,Bombyx mori.BMC Genomics,2010,11:611.
[23]HUANG Q,DEVERAUX Q L,MAEDA S,STENNICKE H R,HAMMOCK B D,REED J C.Cloning and characterization of an inhibitor of apoptosis protein(IAP)from Bombyx mori.Biochimica et Biophysica Acta,2001,1499(3):191-198.
[24]HAMAJIMA R,IWAMOTO A,TOMIZAKI M,SUGANUMA I,KITAGUCHI K,KOBAYASHI M,YAMADA H,IKEDA M.Functional analysis of inhibitor of apoptosis 1 of the silkworm Bombyx mori.Insect Biochemistry and Molecular Biology,2016,79:97-107.
[25]PAN M H,CAI X J,LIU M,LV J,TANG H,TAN J,LU C.Establishment and characterization of an ovarian cell line of the silkworm,Bombyx mori.Tissue and Cell,2010,42(1):42-46.
[26]ZHANG J,HE Q,ZHANG C D,CHEN X Y,CHEN X M,DONG ZQ,LI N,KUANG X X,CAO M Y,LU C,PAN M H.Inhibition of BmNPV replication in silkworm cells using inducible and regulated artificial microRNA precursors targeting the essential viral gene lef-11.Antiviral Research,2014,104:143-152.
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