家蚕微孢子虫极管蛋白2(NbPTP2)的表达、纯化和定位特征
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摘要
【目的】微孢子虫是一种细胞内专性寄生的真核生物,几乎能感染所有生物,包括人类。极管作为微孢子虫特有的侵染器官,主要由极管蛋白组成。极管蛋白在微孢子虫侵染宿主与稳定孢子结构中发挥重要作用。本研究通过克隆、表达家蚕微孢子虫(Nosema bombycis)极管蛋白2(NbPTP2),分析其在成熟孢子极管上的定位特征,为深入研究极管蛋白的功能打下基础。【方法】克隆家蚕微孢子虫NbPTP2。利用Expasy、SignalP 4.1、TMHMM Server V.2.0和NetPhos 3.1 Server等在线软件对NbPTP2的序列特征进行分析,包括氨基酸组成、蛋白质理论分子量、等电点、信号肽、跨膜结构域和磷酸化位点。此外,利用MEGA 7.0软件构建不同种属微孢子虫极管蛋白2的系统进化树。通过克隆NbPTP2,将其与原核表达载体pET32a(+)连接,构建pET32a(+)-NbPTP2重组表达质粒。将测序正确的重组质粒转化到大肠杆菌Rosetta中,IPTG诱导异源表达,经镍柱亲和层析纯化融合蛋白后免疫新西兰大白兔制备多克隆抗体。利用免疫印迹检测NbPTP2在成熟孢子中的表达情况,并通过间接免疫荧光试验(IFA)分析NbPTP2在家蚕微孢子虫成熟孢子中的定位特征。【结果】成功克隆并获得长为834 bp的NbPTP2基因序列,该蛋白编码278个氨基酸残基,理论分子质量为30.9 kD,等电点为9.39,无跨膜结构域,N端存在信号肽,具有潜在的磷酸化修饰位点。系统进化树分析结果显示,家蚕微孢子虫NbPTP2与西方蜜蜂微孢子虫NaPTP2、东方蜜蜂微孢子虫NcPTP2的亲缘关系最近。Western blot结果表明,NbPTP2编码的蛋白质在成熟孢子的孢子总蛋白中有表达,分子量大小约为39 kD。IFA定位特征分析结果显示,NbPTP2能定位于家蚕微孢子虫的整条极管上,证实其为一种极管蛋白。【结论】明确了NbPTP2与其他微孢子虫极管蛋白2的亲缘关系,NbPTP2在成熟家蚕微孢子虫中有表达,且能定位于微孢子虫发芽后的整条极管上。研究结果可为极管的结构解析与极管蛋白功能的研究提供依据。
【Objective】Microsporidia are eukaryotic intracellular obligate parasites that infect almost all organisms, including human. As a special infection organ, the polar tube is mainly composed of polar tube proteins. The polar tube protein plays an important role in microsporidia invasion host and maintaining the structure of polar tube. The objective of this study is to clone and express Nosema bombycis polar tube protein 2(NbPTP2), analyze its localization characteristics in mature spores, and to lay a foundation for further study the function of polar tube proteins.【Method】NbPTP2 was amplified from N. bombycis genome. The amino acid composition, theoretical molecular weight and predicted isoelectric point of NbPTP2 were analyzed by Expasy online software. SignalP 4.1 and TMHMM Server V. 2.0 were used to predict the signal peptide and transmembrane domain of NbPTP2.The phosphorylation site of NbPTP2 was analyzed by NetPhos 3.1 Server. The phylogenetic tree of Nb PTP2 from different microsporidia species was constructed by MEGA 7.0. NbPTP2 was amplified from N. bombycis genome, then ligated with prokaryotic expression vector p ET32 a(+). The correctly sequenced recombinant plasmid was transformed into Escherichia coli Rosetta, and protein expression was heterologous induced by IPTG. The polyclonal antibody of NbPTP2 was prepared by immunizing New Zealand rabbits with the fusion protein by affinity chromatography purification. The expression of NbPTP2 in mature spores was detected by Western blot. Indirect immunofluorescence assay(IFA) was used to analyze the localization characteristics of NbPTP2 in mature spores of microsporidia. 【Result】 The NbPTP2 with a length of 834 bp was successfully cloned. The protein encodes 278 amino acid residues with a theoretical molecular weight of 30.9 kD, and isoelectric point of 9.39.Moreover, it was predicted to have a N-terminal signal peptide and potential phosphoric acid sites, but no transmembrane domain.The phylogenetic tree analysis result showed that NbPTP2 from N. bombycis was closely related to NaPTP2 from N. apis and NcPTP2 from N. ceranae. Western blot result showed that NbPTP2 was expressed in mature spores of N. bombycis and its molecular weight was about 39 kD. The localization analysis result of IFA indicated that NbPTP2 could locate on the whole polar tube of N.bombycis, and it was confirmed that NbPTP2 was a polar tube protein. 【Conclusion】 The relationship between NbPTP2 and polar tube protein 2 from other microsporidia was clarified. NbPTP2 was expressed in N. bombycis and could be localized on the whole polar tube after germination. These results can provide a basis for polar tube structure analysis and polar tube protein function research.
【Objective】Microsporidia are eukaryotic intracellular obligate parasites that infect almost all organisms, including human. As a special infection organ, the polar tube is mainly composed of polar tube proteins. The polar tube protein plays an important role in microsporidia invasion host and maintaining the structure of polar tube. The objective of this study is to clone and express Nosema bombycis polar tube protein 2(NbPTP2), analyze its localization characteristics in mature spores, and to lay a foundation for further study the function of polar tube proteins.【Method】NbPTP2 was amplified from N. bombycis genome. The amino acid composition, theoretical molecular weight and predicted isoelectric point of NbPTP2 were analyzed by Expasy online software. SignalP 4.1 and TMHMM Server V. 2.0 were used to predict the signal peptide and transmembrane domain of NbPTP2.The phosphorylation site of NbPTP2 was analyzed by NetPhos 3.1 Server. The phylogenetic tree of Nb PTP2 from different microsporidia species was constructed by MEGA 7.0. NbPTP2 was amplified from N. bombycis genome, then ligated with prokaryotic expression vector p ET32 a(+). The correctly sequenced recombinant plasmid was transformed into Escherichia coli Rosetta, and protein expression was heterologous induced by IPTG. The polyclonal antibody of NbPTP2 was prepared by immunizing New Zealand rabbits with the fusion protein by affinity chromatography purification. The expression of NbPTP2 in mature spores was detected by Western blot. Indirect immunofluorescence assay(IFA) was used to analyze the localization characteristics of NbPTP2 in mature spores of microsporidia. 【Result】 The NbPTP2 with a length of 834 bp was successfully cloned. The protein encodes 278 amino acid residues with a theoretical molecular weight of 30.9 kD, and isoelectric point of 9.39.Moreover, it was predicted to have a N-terminal signal peptide and potential phosphoric acid sites, but no transmembrane domain.The phylogenetic tree analysis result showed that NbPTP2 from N. bombycis was closely related to NaPTP2 from N. apis and NcPTP2 from N. ceranae. Western blot result showed that NbPTP2 was expressed in mature spores of N. bombycis and its molecular weight was about 39 kD. The localization analysis result of IFA indicated that NbPTP2 could locate on the whole polar tube of N.bombycis, and it was confirmed that NbPTP2 was a polar tube protein. 【Conclusion】 The relationship between NbPTP2 and polar tube protein 2 from other microsporidia was clarified. NbPTP2 was expressed in N. bombycis and could be localized on the whole polar tube after germination. These results can provide a basis for polar tube structure analysis and polar tube protein function research.
引文
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[23]BOUZAHZAH B,WEISS L.Glycosylation of the major polar tube protein of Encephalitozoon cuniculi.Parasitology Research,2010,107(3):761-764.
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[29]吴玉娇,龙梦娴,陈洁,李治,李致宏,潘国庆,李田,周泽扬.家蚕微孢子虫极管蛋白1(NbPTP1)的基因克隆及原核表达.蚕业科学,2014,40(2):258-264.WU Y J,LONG M X,CHEN J,LI Z,LI Z H,PAN G Q,LI T,ZHOUZ Y.Cloning and prokaryotic expression of Nosema bombycis polar tube protein 1(NbPTP1).Science of Sericulture,2014,40(2):258-264.(in Chinese)
[30]龙梦娴,谭瑶瑶,刘柯柯,吴玉娇,吕青,潘国庆,周泽扬.家蚕微孢子虫极管蛋白1(NbPTP1)在果蝇S2细胞中的表达与糖基化修饰.生物工程学报,2018,34(9):1460-1468.LONG M X,TAN Y Y,LIU K K,WU Y J,LüQ,PAN G Q,ZHOU ZY.Expression of polar tube protein 1(NbPTP1)from Nosema bombycis in Drosophila S2 cell lines and its glycosylation.Chinese Journal of Biotechnology,2018,34(9):1460-1468.(in Chinese)
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[2]周泽扬,潘国庆,向仲怀.家蚕微孢子虫研究10年回眸.蚕业科学,2014,40(6):949-956.ZHOU Z Y,PAN G Q,XIANG Z H.A retrospect on researches in Nosema bombycis during the past ten years.Science of Sericulture,2014,40(6):949-956.(in Chinese)
[3]STENTIFORD G D,FEIST S W,STONE D M,BATEMAN K S,DUNN A M.Microsporidia:diverse,dynamic,and emergent pathogens in aquatic systems.Trends in Parasitology,2013,29(11):567-578.
[4]NADIA A E,THOMAS W.Microsporidia.Frontiers in Parasitology,2017,2:221-275.
[5]STENTIFORD G D,BECNEL J J,WEISS L M,KEELING P J,DIDIER E S,WILLIAMS B P,BJORNSON S,KENT M L,FREEMAN M A,BROWN M J F,TROEMEL E R,ROESEL K,SOKOLOVA Y,SNOWDEN K F,SOLTER L.Microsporidiaemergent pathogens in the global food chain.Trends in Parasitology,2016,32(4):336-348.
[6]GORBANZADEH B,SADRAEI J.PP-171 detection of Microsporidia and Cryptosporidium in stool specimens from AIDS patients by modified trichrome-blue and acid-fast trichrome staining methods.International Journal of Infectious Diseases,2011,15(Suppl.1):S93.
[7]TEXIER C,VIDAU C,VIGUèS B,EL ALAOUI H,DELBAC F.Microsporidia:a model for minimal parasite-host interactions.Current Opinion in Microbiology,2010,13(4):443-449.
[8]SZUMOWSKI S C,TROEMEL E R.Microsporidia-host interactions.Current Opinion in Microbiology,2015,26:10-16.
[9]WILLIAMS B A P.Unique physiology of host-parasite interactions in microsporidia infections.Cellular Microbiology,2009,11(11):1551-1560.
[10]BOUZAHZAH B,NAGAJYOTHI F,GHOSH K,TAKVORIAN P M,CALI A,TANOWITZ H B,WEISS L M.Interactions of Encephalitozoon cuniculi polar tube proteins.Infection and Immunity,2010,78(6):2745-2753.
[11]DELBAC F,POLONAIS V.The microsporidian polar tube and its role in invasion.Sub-cellular Biochemistry,2008,47:208-220.
[12]XU Y J,WEISS L M.The microsporidian polar tube:a highly specialised invasion organelle.International Journal for Parasitology,2005,35(9):941-953.
[13]WITTNER M,WEISS L M.The Microsporidia and Microsporidiosis.ASM Press,1999.
[14]POLONAIS V,BELKORCHIA A,ROUSSEL M,PEYRETAILLADEE,PEYRET P,DIOGON M,DELBAC F.Identification of two new polar tube proteins related to polar tube protein 2 in the microsporidian Antonospora locustae.FEMS Microbiology Letters,2013,346(1):36-44.
[15]KEOHANE E M,ORR G A,TAKVORIAN P M,CALI A,TANOWITZ H B,WITTNER M,WEISS L M.Purification and characterization of a microsporidian polar tube protein.Molecular and Biochemical Parasitology,1996,79(2):255-259.
[16]WEIDNER E,BYRD W.The microsporidian spore invasion tube.II.Role of calcium in the activation of invasion tube discharge.The Journal of Cell Biology,1982,93(3):970-975.
[17]高永珍,黄可威,常智杰.家蚕病原性微孢子虫极丝蛋白的研究.蚕业科学,2001,27(2):128-130.GAO Y Z,HUANG K W,CHANG Z J.Studies on the polar tube proteins of microsporidium pathogenic to silkworm.Science of Sericulture,2001,27(2):128-130.(in Chinese)
[18]PEUVEL I,PEYRET P,MéTéNIER G,VIVARèS C P,DELBAC F.The microsporidian polar tube:evidence for a third polar tube protein(PTP3)in Encephalitozoon cuniculi.Molecular and Biochemical Parasitology,2002,122(1):69-80.
[19]DELBAC F,PEYRET P,METENIER G,DAVID D,DANCHIN A,VIVARèS C P.On proteins of the microsporidian invasive apparatus:complete sequence of a polar tube protein of Encephalitozoon cuniculi.Molecular Microbiology,1998,29(3):825-834.
[20]WEISS L M,DELBAC F,RUSSELL HAYMAN J,PAN G Q,DANGX Q,ZHOU Z Y.The microsporidian polar tube and spore wall//Microsporidia:Pathogens of Opportunity,2014:261-306.
[21]POLONAIS V.Identification of new polar tube and spore wall proteins in different microsporidian species.First attempts for the transfection of the microsporidia Encephalitozoon cuniculi[D].France:UniversitéBlaise Pascal-Clermont-Ferrand II,2006.
[22]SINSUWONKWAT W,SANYATHITISEREE P,PHATTANAKUNANANS,JALA S,LERTWATCHARASARAKUL P.Optimized codons of polar tube protein 1 gene of Encephalitozoon cuniculi to enhance protein expression in Escherichia coli.Thai Journal of Veterinary Medicine,2016,46(4):579-587.
[23]BOUZAHZAH B,WEISS L.Glycosylation of the major polar tube protein of Encephalitozoon cuniculi.Parasitology Research,2010,107(3):761-764.
[24]WEIDNER E.The microsporidian spore invasion tube.The ultrastructure,isolation,and characterization of the protein comprising the tube.The Journal of Cell Biology,1976,71(1):23-34.
[25]POLONAIS V,PRENSIER G,MéTéNIER G,VIVARèS C P,DELBAC F.Microsporidian polar tube proteins:Highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups.Fungal Genetics and Biology,2005,42(9):791-803.
[26]HAN B,POLONAIS V,SUGI T,YAKUBU R,TAKVORIAN P M,CALI A,MAIER K,LONG M X,LEVY M,TANOWITZ H B,PANG,DELBAC F,ZHOU Z,WEISS L M.The role of microsporidian polar tube protein 4(PTP4)in host cell infection.PLoS Pathogens,2017,13(4):e1006341.
[27]BROSSON D,KUHN L,DELBAC F,GARIN J,VIVARèS C P,TEXIER C.Proteomic analysis of the eukaryotic parasite Encephalitozoon cuniculi(microsporidia):a reference map for proteins expressed in late sporogonial stages.Proteomics,2006,6(12):3625-3635.
[28]PAN G,XU J,LI T,XIA Q Y,LIU S L,ZHANG G J,LI S G,LI C F,LIU H D,YANG L,et al.Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation.BMC Genomics,2013,14:186.
[29]吴玉娇,龙梦娴,陈洁,李治,李致宏,潘国庆,李田,周泽扬.家蚕微孢子虫极管蛋白1(NbPTP1)的基因克隆及原核表达.蚕业科学,2014,40(2):258-264.WU Y J,LONG M X,CHEN J,LI Z,LI Z H,PAN G Q,LI T,ZHOUZ Y.Cloning and prokaryotic expression of Nosema bombycis polar tube protein 1(NbPTP1).Science of Sericulture,2014,40(2):258-264.(in Chinese)
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