赵焕之;赵其平;朱顺海;王璐;刘桂玲;李志行;董辉;黄兵;韩红玉;

• 1:中国农业科学院上海兽医研究所农业农村部动物寄生虫学重点开放实验室
• 2:上海师范大学生命与环境科学学院

摘要(Abstract)：

为探究柔嫩艾美耳球虫钙依赖蛋白激酶3(EtCDPK3)在钙离子信号通路中是如何将信号传递给下游效应分子,进一步促进虫体入侵宿主细胞的机制,以柔嫩艾美耳球虫子孢子蛋白及其单克隆抗体和多克隆抗体为实验材料,利用免疫共沉淀技术筛选EtCDPK3的互作蛋白并进行质谱鉴定分析,使用Maxquant软件对获得的原始数据进行分析。SDS-PAGE结果显示,与阴性对照组相比,2个样品组出现差异条带;免疫共沉淀洗脱液质谱鉴定分析结果显示,获得6个可能与EtCDPK3相互作用的相关蛋白。以上实验结果为进一步深入研究EtCDPK3在柔嫩艾美耳球虫入侵宿主细胞过程中的作用奠定基础。

关键词(KeyWords)： 柔嫩艾美耳球虫;钙依赖蛋白激酶3;免疫共沉淀;质谱;互作蛋白

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31572266);; 国家寄生虫种质资源共享服务平台(平台-TDRC-22);; 上海市闵行区人才发展专项基金

作者(Author): 赵焕之;赵其平;朱顺海;王璐;刘桂玲;李志行;董辉;黄兵;韩红玉;

Email:

DOI:

参考文献(References)：

• [1]Harmon A C,Putnam-Evans C,Cormier M J.A calcium-dependent but calmodulin-independent protein kinase from soybean[J].Plant Physiol,1987,83(4):830-837.
• [2]Harper J F,Sussman M R,Schaller G E,et al.A calcium-dependent protein kinase with a regulatory domain similar to calmodulin[J].Science,1991,252(5008):951-954.
• [3]Yuasa T,Muto S.Ca(2+)-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta:partial purification and Ca(2+)-dependent association of the enzyme to the microsomes[J].Arch Biochem Biophys,1992,296(1):175-182.
• [4]陈硕,陈珈.植物中钙依赖蛋白激酶(CDPKs)的结构与功能[J].植物学通报,2001,18(2):143-148.
• [5]麻浩,王爽,周亚丽.植物中钙依赖蛋白激酶的研究进展[J].南京农业大学学报,2017,40(4):565-572.
• [6]Phee B K,Shin D H,Cho J H,et al.Identification of phytochrome-interacting protein candidates in Arabidopsis thaliana by co-immunoprecipitation coupled with MALDI-TOF MS[J].Proteomics,2006,6(12):3671-3680.
• [7]Free R B,Hazelwood L A,Sibley D R.Identifying novel protein-protein interactions using co-immunoprecipitation and mass spectroscopy[J].Curr Protoc Neurosci,2009,Chapter 5:Unit 5.28.
• [8]Huang B X,Kim H Y.Effective identification of Akt interacting proteins by two-step chemical crosslinking,co-immunoprecipitation and mass spectrometry[J].PLoS One,2013,8(4):e61430.
• [9]张頔,赵魁,周艳龙,等.免疫共沉淀联合质谱技术筛选宿主因子CypB的互作蛋白[J].中国兽医学报,2017,37(5):905-908.
• [10]刘昆,左兆云,贾慧娜,等.免疫共沉淀联合质谱技术筛选绵羊α-生育酚转移蛋白的互作蛋白[J].中国畜牧杂志,2014,50(15):80-83.
• [11]王成,赵晓蒙,帅勇,等.免疫共沉淀结合质谱分析筛选HMGB4相互作用蛋白[J].湖南师范大学自然科学学报,2012,35(2):71-75.
• [12]李耀东,关振宏,严景华.免疫共沉淀筛选细胞内与A型流感病毒M2蛋白相互作用的蛋白[J].微生物学报,2009,49(8):1081-1085.
• [13]李洋,韩红玉,黄兵.顶复器原虫钙依赖蛋白激酶的研究进展[J].热带医学杂志,2010,10(2):223-225,231.
• [14]张念章,陈佳,王萌,等.顶复门原虫钙依赖蛋白激酶的研究进展[J].畜牧兽医学报,2013,44(1):1-6.
• [15]Siden-Kiamos I,Ecker A,Nyb?ck S,et al.Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion[J].Mol Microbiol,2006,60(6):1355-1363.
• [16]Gaji R Y,Johnson D E,Treeck M,et al.Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress[J].PLoS Pathog,2015,11(11):e1005268.
• [17]Han H Y,Zhu S H,Jiang L L,et al.Molecular characterization and analysis of a novel calcium-dependent protein kinase from Eimeria tenella[J].Parasitology,2013,140(6):746-755.
• [18]Wang Z,Huang B,Dong H,et al.Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella[J].PLoS One,2016,11(12):e0168132.
• [19]Lv L,Huang B,Zhao Q,et al.Identification of an interaction between calcium-dependent protein kinase 4 (EtCDPK4) and serine protease inhibitor (EtSerpin) in Eimeria tenella[J].Parasit Vectors,2018,11(1):259.
• [20]阎晓菲,孔苗,韩红玉,等.堆型艾美耳球虫ATP酶基因的生物信息学分析及原核表达[J].中国动物传染病学报,2018,27(3):65-73.
• [21]陈婷,黄兵,赵其平,等.柔嫩艾美耳球虫伴侣蛋白TCP-1亚基α的特性和功能初步分析[J].中国动物传染病学报,2018,26(2):40-48.
• [22]Rahal R.Analysis of Protein-Protein Interactions Between MEN Components[J].Methods Mol Biol,2017 (1505):81-88.
• [23]Ray N B,Mathews C K.Nucleoside diphosphokinase:a functional link between intermediary metabolism and nucleic acid synthesis[J].Curr Top Cell Regul,1992(33):343-357.
• [24]Lu Q,Park H,Egger L A.Nucleoside-diphosphate kinase-mediated signal transduction via histidyl-aspartyl phosphorelay systems in Escherichia coli[J].J Biol Chem,1996,271(51):32886-32893.
• [25]Kumar P,Verma A,Saini AK,et al.Nucleoside diphosphate kinase from Mycobacterium tuberculosis cleaves single strand DNA within the human c-myc promoter in an enzyme-catalyzed reaction[J].Nucleic Acids Res,2005,33(8):2707-2714.
• [26]Yu H,Xiong J,Zhang R,et al.Ndk,a novel host-responsive regulator,negatively regulates bacterial virulence through quorum sensing in Pseudomonas aeruginosa[J].Sci Rep,2016(6):28684.
• [27]Marr J J.Purine analogs as chemotherapeutic agents in leishmaniasis and American trypanosomiasis[J].J Lab Clin Med,1991,118(2):111-119.
• [28]Kolli B K,Kostal J,Zaborina O,et al.Leishmania-released nucleoside diphosphate kinase prevents ATP-mediated cytolysis of macrophages[J].Mol Biochem Parasitol,2008,158(2):163-175.
• [29]Vieira P S,Souza TACB,Honorato R V,et al.Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites[J].Biochem Biophys Res Commun,2017,488(3):461-465.
• [30]Shkolnik K,Ben-Dor S,Galiani D,et al.Molecular characterization and bioinformatics analysis of Ncoa7B,a novel ovulation-associated and reproduction system-specific Ncoa7 isoform[J].Comparative Study,2008,135(3):321-333.
• [31]Beitner R,Nordenberg J.Inhibition of 6-phosphoglu-conate dehydrogenase (decarboxylating) by glucose 1,6-bisphosphate[J].Biochim Biophys Acta,1979,583(2):266-269.
• [32]Pascual C,Herrera L S.Coordinate regulation of the pentose phosphate pathway and of the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (decarboxylating)[J].Folia Microbiol (Praha),1982,27(6):365-369.
• [33]He W,Wang Y,Liu W,et al.Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1[J].BMC Struct Biol,2007(7):38.
• [34]Esparza-MoltóP B,Nuevo-Tapioles C,Cuezva J M.Regulation of the H+-ATP synthase by IF1:a role in mitohormesis[J].Cell Mol Life Sci,2017,74(12):2151-2166.
• [35]Long Q,Yang K,Yang Q.Regulation of mitochondrial ATP synthase in cardiac pathophysiology[J].Am J Cardiovasc Dis,2015,5(1):19-32.
• [36]关丫丫,章小英,王辉,等.线粒体ATP合酶抑制因子(ATPIF1)在能量代谢中的作用[J].生理科学进展,2018,49(3):230-236.
• [37]Beamer L J.Mutations in hereditary phosphoglucomutase 1 deficiency map to key regions of enzyme structure and function[J].J Inherit Metab Dis,2015,38(2):243-256.
• [38]Stiers K M,Kain B N,Graham A C,et al.Induced Structural Disorder as a Molecular Mechanism for Enzyme Dysfunction in Phosphoglucomutase 1 Deficiency[J].J Mol Biol,2016,428(8):1493-1505.