我国羊巴贝斯虫棒状体颈部蛋白RON2基因的克隆与结构和遗传进化分析
|
摘要
用牛巴贝斯虫棒状体颈部蛋白RON2基因序列BLAST我国羊感染的2种巴贝斯虫全基因组数据库,以获得的RON2基因保守序列片段为模板设计PCR引物;从6株羊巴贝斯虫基因组DNA和c DNA中扩增RON2基因全长序列,分析其基因结构特征;并通过序列比对和进化树构建,分析我国羊巴贝斯虫的遗传进化关系。结果显示,该基因无内含子,羊巴贝斯虫未定种新疆株/敦煌株(BspX J/DH)、莫氏巴贝斯虫天祝株/临潭株(BmTZ/LT)和莫氏巴贝斯虫宁县株/河北株(BmNX/HB)RON2蛋白基因ORF的大小分别为4 029、4 047和4 044 bp,编码1 345、1 349和1 348个氨基酸。RON2蛋白的分子质量约为151 ku,等电点为8.91,具有3个跨膜区,N端第1～29位氨基酸为其信号肽区域。RON2核苷酸和氨基酸序列比对分析结果显示,BspXJ/DH与BmTZ/LT和BmNX/HB的核苷酸和氨基酸序列相似性分别为67.0%～67.4%和69.2%～69.3%,BmTZ/LT和BmNX/HB间的相似性分别为92.8%～92.9%和91.1%～91.2%。系统发生树上羊巴贝斯虫未定种和莫氏巴贝斯虫分别位于2大支,莫氏巴贝斯虫又被分为2小支。该结果为开展我国羊巴贝斯虫的分类关系和RON2基因功能的研究提供了基础数据。
The conserved sequence fragments were obtained using rhoptry neck protein 2(RON2) gene of Babesia bovis to BLAST local genomic databases of two Babesia species infective to sheep and goats in China. The primers of PCR were designed using the fragment sequences as templates. The full-length RON2 gene sequences of 6 Chinese ovine Babesia strains were amplified from genomic DNAs and c DNAs,and these gene structural characteristics were analyzed. The genetic evolutionary relationship between these ovine Babesia strains was analyzed via sequence alignment and construction of phylogenetic trees. The result showed that there was not intron in the RON2 gene of these Babesia strains. The ORFs of Babesia sp.Xinjiang/Dunhuang(Bsp XJ/DH),B.motasi Tianzhu/Lintan(Bm TZ/LT) and B.motasi Ningxian/Hebei(BmNX/HB) RON2 gene were 4 029 bp,4 047 bp and 4 044 bp in length,and encoded 1 345,1 349 and 1 348 amino acids,respectively.The RON2 proteins of these ovine Babesia strains were about 151 ku in molecular weight and their isoelectric points were 8.91.This protein had three transmembrane domains. The initiative 29 amino acids in N terminal were the signal peptide.The alignment of RON2 nucleotide and amino acid sequences indicated that the similarities of Bsp XJ/DH and strains of B.motasi were 67.0%—67.4%and 69.2%—69.3%,and that between Bm TZ/LT and Bm NX/HB was 92.8%—92.9% and 91.1%—91.2%,respectively.The 6 ovine Babesia strains were located on 2 clades,and 4 strains of B.motasi were further divided into 2 sub-clades in phylogenetic tree.These results provided the foundational data for further exploring the genetic evolution relationship of these ovine Babesia strains and the function of Babesia RON2 gene.
The conserved sequence fragments were obtained using rhoptry neck protein 2(RON2) gene of Babesia bovis to BLAST local genomic databases of two Babesia species infective to sheep and goats in China. The primers of PCR were designed using the fragment sequences as templates. The full-length RON2 gene sequences of 6 Chinese ovine Babesia strains were amplified from genomic DNAs and c DNAs,and these gene structural characteristics were analyzed. The genetic evolutionary relationship between these ovine Babesia strains was analyzed via sequence alignment and construction of phylogenetic trees. The result showed that there was not intron in the RON2 gene of these Babesia strains. The ORFs of Babesia sp.Xinjiang/Dunhuang(Bsp XJ/DH),B.motasi Tianzhu/Lintan(Bm TZ/LT) and B.motasi Ningxian/Hebei(BmNX/HB) RON2 gene were 4 029 bp,4 047 bp and 4 044 bp in length,and encoded 1 345,1 349 and 1 348 amino acids,respectively.The RON2 proteins of these ovine Babesia strains were about 151 ku in molecular weight and their isoelectric points were 8.91.This protein had three transmembrane domains. The initiative 29 amino acids in N terminal were the signal peptide.The alignment of RON2 nucleotide and amino acid sequences indicated that the similarities of Bsp XJ/DH and strains of B.motasi were 67.0%—67.4%and 69.2%—69.3%,and that between Bm TZ/LT and Bm NX/HB was 92.8%—92.9% and 91.1%—91.2%,respectively.The 6 ovine Babesia strains were located on 2 clades,and 4 strains of B.motasi were further divided into 2 sub-clades in phylogenetic tree.These results provided the foundational data for further exploring the genetic evolution relationship of these ovine Babesia strains and the function of Babesia RON2 gene.
引文
[1] ROS-GARCA A,BARANDIKA J F,GARCA-PREZ A L,et al.Assessment of exposure to piroplasms in sheep grazing in communal mountain pastures by using a multiplex DNA bead-based suspension array[J].Parasites&Vectors,2013,6(1):277.
[2] HASSAN M A,LIU Jun-long,RASHID M,et al.Molecular survey of piroplasm species from selected areas of China and Pakistan[J].Parasites&Vectors,2018,11(1):457.
[3] GUAN Gui-quan,CHAUVIN A,LUO Jian-xun,et al.The development and evaluation of a loop-mediated isothermal amplification(LAMP)method for detection of Babesia spp. infective to sheep and goats in China[J].Experimental Parasitology,2008,120(1):39-44.
[4] GUAN Gui-quan,CHAUVIN A,ROGNIAUX H,et al.Merozoite proteins from Babesia sp.BQ1(Lintan)as potential antigens for serodiagnosis by ELISA[J].Parasitology,2010,137(6):927-938.
[5] GUAN Gui-quan,MA Mi-ling,LIU Ai-hong,et al. A recently identified ovine Babesia in China:Serology and sero-epidemiology[J].Parasitology International,2012,61(4):532-537.
[6]杨强,刘爱红,刘军龙,等.我国十省份羊巴贝斯虫的分子流行病学调查[J].中国兽医科学,2016,46(5):597-601.YANG Qiang,LIU Ai-hong,LIU Jun-long,et al.Molecular epidemiological investigation of ovine Babesia spp.in 10 provinces of China[J].Chinese Veterinary Science,2016,46(5):597-601.(in Chinese)
[7] DUBREMETZ J F,GARCIA-RGUET N,CONSEIL V,et al.Apical organelles and host-cell invasion by Apicomplexa[J].International Journal for Parasitology,1998,28(7):1007-1013.
[8] SANTOS J M,FERGUSON D J P,BLACKMAN M J,et al.Intramembrane cleavage of AMA1 triggers Toxoplasma to switch from an invasive to a replicative mode[J].Science,2011,331(6016):473-477.
[9] ALEXANDER D L,MITAL J,WARD G E,et al.Identification of the moving junction complex of Toxoplasma gondii:A collaboration between distinct secretory organelles[J].PLoS Pathogens,2005,1(2):e17.
[10] AIKAWA M,MILLER L H,JOHNSON J,et al.Erythrocyte entry by malarial parasites. A moving junction between erythrocyte and parasite[J].Journal of Cell Biology,1978,77(1):72-82.
[11] RICHARD D,MACRAILD C,RIGLAR D T,et al.Interaction between Plasmodium falciparum apical membrane antigen 1 and the rhoptry neck protein complex defines a key step in the erythrocyte invasion process of malaria parasites[J].Journal of Biological Chemistry,2010,285(19):14815-14822.
[12] IGONET S,VULLIEZ-LE BNORMAND B,FAURE G,et al.Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody:binding and structural characterisation[J].Journal of Molecular Biology,2007,366(5):1523-1537.
[13] BEGHETTO E,NIELSEN H V,DEL PORTO P,et al.A combination of antigenic regions of Toxoplasma gondii microneme proteins induces protective immunity against oral infection with parasite cysts[J].Journal of Infectious Diseases,2005,191(4):637-644.
[14] GOU Hui-tian,GUAN Gui-quan,MA Mi-ling,et al.Phylogenetic analysis based on 28S rRNA of Babesia spp. in ruminants in China[J].Experimental&Applied Acarology,2013,59(4):463-472.
[15] TIAN Zhan-cheng,LIU Guang-yuan,YIN Hong,et al.RPS8-a new informative DNA marker for phylogeny of Babesia and Theileria parasites in China[J].PLoS One,2013,8(11):e79860.
[16] NIU Qing-li,LUO Jian-xun,GUAN Gui-quan,et al.Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer(ITS)sequences[J].Experimental Parasitology,2009,121(1):64-68.
[17] GUAN Gui-quan,KORHONEN P K,YOUNG N D,et al.Genomic resources for a unique,low-virulence Babesia taxon from China[J].Parasites&Vectors,2016,9(1):564.
[18]何欣,刘军龙,刘爱红,等.我国羊巴贝斯虫trap基因结构及遗传进化分析[J].畜牧兽医学报,2017,48(7):1332-1341.HE Xin,LIU Jun-long,LIU Ai-hong,et al.The structural and phylogenetic analysis of trap gene in ovine Babesia species in China[J].Acta Veterinaria et Zootechnica Sinica,2017,48(7):1332-1341.(in Chinese)
[19] SAMYELLOWE T Y.Rhoptry organelles of the Apicomplexa:Their role in host cell invasion and intracellular survival[J].Parasitology Today,1996,12(8):308-316.
[20] KANEKO O,TSUBOI T,LING I T,et al.The high molecular mass rhoptry protein,RhopH1,is encoded by members of the clag multigene family in Plasmodium falciparum and Plasmodium yoelii[J].Molecular&Biochemical Parasitology,2001,118(2):223-231.
[21] BRADLEY P J,WARD C,CHENG S J,et al.Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii[J].The Journal of Biological Chemistry,2005,280(40):34245-34258.
[22] LAMARQUE M,BESTEIRO S,PAPOIN J,et al.The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by Apicomplexan parasites[J].PLoS Pathogens,2011,7(2):e1001276.
[23] ALEXANDER D L,ARASTU-KAPUR S,DUBREMETZ J F,et al.Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4,a component of the moving junction in Toxoplasma gondii[J].Eukaryotic Cell,2006,5(7):1169-1173.
[24] BESTEIRO S,MICHELIN A,PONCET J,et al.Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion[J].PLo S Pathogens,2009,5(2):e1000309.
[25] TYLER J S,BOOTHROYD J C.The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex[J]. PLoS Pathogens,2011,7(2):e1001282.
[26] CAO J,KANEKO O,THONGKUKIATKUL A,et al.Rhoptry neck protein RON2 forms a complex with microneme protein AMA1 in Plasmodium falciparum merozoites[J].Parasitology International,2009,58(1):29-35.
[27] COLLINS W E,WALDUCK A,SULLIVAN J S,et al.Efficacy of vaccines containing rhoptry-associated proteins RAP1 and RAP2 of Plasmodium falciparum in Saimiri boliviensis monkeys[J].American Journal of Tropical Medicine&Hygiene,2000,62(4):466.
[28] STRAUB K W,CHENG S J,SOHN C S,et al.Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements[J].Cellular Microbiology,2010,11(4):590-603.
[29] GARCIA J L,GENNARI S M,NAVARRO I T,et al.Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii[J].Veterinary Parasitology,2005,129(3):209-217.
[30] ARVALO-PINZN G,CURTIDOR H,PATIO L C,et al.PvRON2,a new Plasmodium vivax rhoptry neck antigen[J].Malaria Journal,2011,10(1):60.
[2] HASSAN M A,LIU Jun-long,RASHID M,et al.Molecular survey of piroplasm species from selected areas of China and Pakistan[J].Parasites&Vectors,2018,11(1):457.
[3] GUAN Gui-quan,CHAUVIN A,LUO Jian-xun,et al.The development and evaluation of a loop-mediated isothermal amplification(LAMP)method for detection of Babesia spp. infective to sheep and goats in China[J].Experimental Parasitology,2008,120(1):39-44.
[4] GUAN Gui-quan,CHAUVIN A,ROGNIAUX H,et al.Merozoite proteins from Babesia sp.BQ1(Lintan)as potential antigens for serodiagnosis by ELISA[J].Parasitology,2010,137(6):927-938.
[5] GUAN Gui-quan,MA Mi-ling,LIU Ai-hong,et al. A recently identified ovine Babesia in China:Serology and sero-epidemiology[J].Parasitology International,2012,61(4):532-537.
[6]杨强,刘爱红,刘军龙,等.我国十省份羊巴贝斯虫的分子流行病学调查[J].中国兽医科学,2016,46(5):597-601.YANG Qiang,LIU Ai-hong,LIU Jun-long,et al.Molecular epidemiological investigation of ovine Babesia spp.in 10 provinces of China[J].Chinese Veterinary Science,2016,46(5):597-601.(in Chinese)
[7] DUBREMETZ J F,GARCIA-RGUET N,CONSEIL V,et al.Apical organelles and host-cell invasion by Apicomplexa[J].International Journal for Parasitology,1998,28(7):1007-1013.
[8] SANTOS J M,FERGUSON D J P,BLACKMAN M J,et al.Intramembrane cleavage of AMA1 triggers Toxoplasma to switch from an invasive to a replicative mode[J].Science,2011,331(6016):473-477.
[9] ALEXANDER D L,MITAL J,WARD G E,et al.Identification of the moving junction complex of Toxoplasma gondii:A collaboration between distinct secretory organelles[J].PLoS Pathogens,2005,1(2):e17.
[10] AIKAWA M,MILLER L H,JOHNSON J,et al.Erythrocyte entry by malarial parasites. A moving junction between erythrocyte and parasite[J].Journal of Cell Biology,1978,77(1):72-82.
[11] RICHARD D,MACRAILD C,RIGLAR D T,et al.Interaction between Plasmodium falciparum apical membrane antigen 1 and the rhoptry neck protein complex defines a key step in the erythrocyte invasion process of malaria parasites[J].Journal of Biological Chemistry,2010,285(19):14815-14822.
[12] IGONET S,VULLIEZ-LE BNORMAND B,FAURE G,et al.Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody:binding and structural characterisation[J].Journal of Molecular Biology,2007,366(5):1523-1537.
[13] BEGHETTO E,NIELSEN H V,DEL PORTO P,et al.A combination of antigenic regions of Toxoplasma gondii microneme proteins induces protective immunity against oral infection with parasite cysts[J].Journal of Infectious Diseases,2005,191(4):637-644.
[14] GOU Hui-tian,GUAN Gui-quan,MA Mi-ling,et al.Phylogenetic analysis based on 28S rRNA of Babesia spp. in ruminants in China[J].Experimental&Applied Acarology,2013,59(4):463-472.
[15] TIAN Zhan-cheng,LIU Guang-yuan,YIN Hong,et al.RPS8-a new informative DNA marker for phylogeny of Babesia and Theileria parasites in China[J].PLoS One,2013,8(11):e79860.
[16] NIU Qing-li,LUO Jian-xun,GUAN Gui-quan,et al.Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer(ITS)sequences[J].Experimental Parasitology,2009,121(1):64-68.
[17] GUAN Gui-quan,KORHONEN P K,YOUNG N D,et al.Genomic resources for a unique,low-virulence Babesia taxon from China[J].Parasites&Vectors,2016,9(1):564.
[18]何欣,刘军龙,刘爱红,等.我国羊巴贝斯虫trap基因结构及遗传进化分析[J].畜牧兽医学报,2017,48(7):1332-1341.HE Xin,LIU Jun-long,LIU Ai-hong,et al.The structural and phylogenetic analysis of trap gene in ovine Babesia species in China[J].Acta Veterinaria et Zootechnica Sinica,2017,48(7):1332-1341.(in Chinese)
[19] SAMYELLOWE T Y.Rhoptry organelles of the Apicomplexa:Their role in host cell invasion and intracellular survival[J].Parasitology Today,1996,12(8):308-316.
[20] KANEKO O,TSUBOI T,LING I T,et al.The high molecular mass rhoptry protein,RhopH1,is encoded by members of the clag multigene family in Plasmodium falciparum and Plasmodium yoelii[J].Molecular&Biochemical Parasitology,2001,118(2):223-231.
[21] BRADLEY P J,WARD C,CHENG S J,et al.Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii[J].The Journal of Biological Chemistry,2005,280(40):34245-34258.
[22] LAMARQUE M,BESTEIRO S,PAPOIN J,et al.The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by Apicomplexan parasites[J].PLoS Pathogens,2011,7(2):e1001276.
[23] ALEXANDER D L,ARASTU-KAPUR S,DUBREMETZ J F,et al.Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4,a component of the moving junction in Toxoplasma gondii[J].Eukaryotic Cell,2006,5(7):1169-1173.
[24] BESTEIRO S,MICHELIN A,PONCET J,et al.Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion[J].PLo S Pathogens,2009,5(2):e1000309.
[25] TYLER J S,BOOTHROYD J C.The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex[J]. PLoS Pathogens,2011,7(2):e1001282.
[26] CAO J,KANEKO O,THONGKUKIATKUL A,et al.Rhoptry neck protein RON2 forms a complex with microneme protein AMA1 in Plasmodium falciparum merozoites[J].Parasitology International,2009,58(1):29-35.
[27] COLLINS W E,WALDUCK A,SULLIVAN J S,et al.Efficacy of vaccines containing rhoptry-associated proteins RAP1 and RAP2 of Plasmodium falciparum in Saimiri boliviensis monkeys[J].American Journal of Tropical Medicine&Hygiene,2000,62(4):466.
[28] STRAUB K W,CHENG S J,SOHN C S,et al.Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements[J].Cellular Microbiology,2010,11(4):590-603.
[29] GARCIA J L,GENNARI S M,NAVARRO I T,et al.Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii[J].Veterinary Parasitology,2005,129(3):209-217.
[30] ARVALO-PINZN G,CURTIDOR H,PATIO L C,et al.PvRON2,a new Plasmodium vivax rhoptry neck antigen[J].Malaria Journal,2011,10(1):60.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |