莫氏巴贝斯虫裂殖子cDNA表达文库的构建及免疫学筛选

摘要/Abstract

摘要：

为了构建莫氏巴贝斯虫（Babesia motasi）裂殖子cDNA表达文库，从中筛选和鉴定功能基因，利用差速离心法从莫氏巴贝斯虫感染的红细胞中纯化裂殖子，提取总RNA并纯化mRNA。在合成的双链cDNA两端加上含EcoRⅠ和Hind Ⅲ定向接头后连接到λ SCREEN载体上。通过体外包装形成完整的噬菌体颗粒，并用之转染ER1647，构建莫氏巴贝斯虫裂殖子的cDNA表达文库。用莫氏巴贝斯虫阳性血清筛选cDNA文库，获得的阳性克隆，经过测序和Blast软件分析鉴定，然后利用末端快速扩增技术（RACE）对筛选到的功能基因片段进行全长扩增。结果表明构建的cDNA表达文库其初级库容量约为1.0×10⁶ PFU，扩增文库为3.5×10⁹ PFU；通过免疫学筛选，获得50个阳性克隆。测序和Blast分析后，鉴定出10个莫氏巴贝斯虫基因片段，RACE扩增获得了其中8个基因的全长。本试验构建的莫氏巴贝斯虫裂殖子的cDNA表达文库和筛选到的10个结构和功能基因，为研究巴贝斯虫生物学特性、筛选疫苗与诊断用抗原及药物靶位奠定了基础。

Abstract:

The objective of this study was to obtain functional genes of Babesia motasi, a cDNA expression library of the merozoites was constructed and immunoscreened with positive sera from sheep infected with B. motasi. The merozoites of B. motasi were purified from red blood cell with differential centrifugation. The mRNA was purified from extracted total RNA. Synthetized double-strand cDNA was added directional EcoRⅠ/Hind Ⅲ linkers and ligated to the EcoRⅠ/HindⅢ arms of λ screen vector. To produce a primary cDNA library of B. motasi, the phages DNA was packaged in vitro and transfected into ER1647. The positive clones were obtained by immunoscreening with the positive sera against B. motasi and amplified with rapid amplification of cDNA ends (RACE). The titers of the primary and amplified cDNA expression library were 1.0×10⁶PFU and 3.5×10⁹ PFU·mL^-1, respectively. The results showed that 10 genes of B. motasi were identified and the full-length of 8 genes were amplified by RACE. The cDNA expression library and genes screened provide an important material for screening and identifying candidate antigens of vaccination and diagnosis, drug targets as well studying biological characteristics of Babesia.

中图分类号:

S852.723

王锦明，刘军龙，刘爱红，马米玲，牛庆丽，任巧云，杨吉飞，刘志杰，李有全，罗建勋，殷宏，关贵全. 莫氏巴贝斯虫裂殖子cDNA表达文库的构建及免疫学筛选[J]. 畜牧兽医学报, doi: .

WANG Jin-ming, LIU Jun-long, LIU Ai-hong, MA Mi-ling, NIU Qing-li, REN Qiao-yun, YANG Ji-fei, LIU Zhi-jie, LI You-quan, LUO Jian-xun, YIN Hong, GUAN Gui-quan. Construction and Immunoscreening of a Merozoites cDNA Expression Library of Babesia motasi[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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参考文献

［1］陈德明. 绵羊焦虫病调查报告［J］. 兽医科技杂志, 1982, (03): 33-34.

［2］赵锡荣, 李翠茹, 闵友贵，等. 绵羊巴贝斯焦虫病的调查［J］. 中国兽医科技, 1986，(01): 28-29.

［3］YIN H, LU W S, LUO J X. Babesiosis in China［J］. Trop Anim Health Pro, 1997, 29(4): 11s-15s.

［4］NIU Q, LUO J, GUAN G Q, et al. Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences［J］. Exp Parasitol, 2009, 121(1): 64-68.

［5］LIU A H, YIN H, GUAN G Q, et al. At least two genetically distinct large Babesia species infective to sheep and goats in China［J］. Vet Parasitol, 2007, 147(3-4): 246-251.

［6］GUAN G Q, YIN H, LUO J X, et al. Transmission of Babesia sp to sheep with field-collected Haemaphysalis qinghaiensis［J］. Parasitol Res, 2002, 88(13 Suppl 1): S22-24.

［7］GUAN G Q, MA M L, MOREAU E, et al. A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum［J］. Exp Parasitol, 2009, 122 (4): 261-267.

［8］GUAN G Q, CHAUVIN A, LUO J X, 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］. Exp Parasitol, 2008, 120(1): 39-44.

［9］GUAN G Q, 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.

［10］GUAN G Q, MA M L , LIU A H, et al. A recently identified ovine Babesia in China: Serology and sero-epidemiology［J］. Parasitol Int, 2012, 61(4):532-537.

［11］NIU Q L, LUO J X, GUAN G Q, et al. Detection and differentiation of ovine Theileria and Babesia by reverse line blotting in China［J］. Parasitology Research, 2009, 104(6): 1417-1423.

［12］SIM G K, KAFATOS F C, JONES C W, et al. Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families［J］. Cell, 1979, 18(4): 1303-1316.

［13］王燕妮. 恶性疟原虫(海南株)CDNA表达文库的构建及鉴定［J］.中国寄生虫学与寄生虫病杂志，1997，(1): 21-25.

［14］吴翔, 蒋立平, 舒衡平，等. 抗弓形虫单克隆抗体免疫筛选弓形虫速殖子cDNA文库［J］. 中国人兽共患病杂志, 2004, (04): 288-290.

［15］王丹静, 蔡力汀, 蒋立平，等. 免疫筛选弓形虫速殖子cDNA文库［J］. 中国寄生虫病防治杂志, 2004, (01): 7-10.

［16］杨秋林, 陆惠民, 邬敏辰，等. 弓形虫昆山分离株速殖子期表达型cDNA文库的构建及鉴定［J］. 中国人兽共患病杂志, 2000, (05): 25-27.

［17］王建民, 张伟, 陈通，等. 鸡柔嫩艾美耳球虫cDNA文库构建与表达序列标签分析［J］. 生物医学工程学杂志, 2007, (06): 1357-1362.

［18］李有全, 罗建勋, 高金亮, 等. 吕氏泰勒虫cDNA表达文库的构建及其抗原基因的免疫筛选［J］. 中国农业科学, 2009, (01): 331-339.

［19］陈启军，李德昌，闫仲堂. 吉氏巴贝斯虫cDNA文库的构建［J］. 中国兽医学报, 1995, (01): 38-42.

［20］RUEF B J, DOWLING S C, CONLEY P G, et al. A unique Babesia bovis spherical body protein is conserved among geographic isolates and localizes to the infected erythrocyte membrane［J］. Mol Biochem Parasitol, 2000, 105(1): 1-12.

［21］刘琴. 东方巴贝斯虫cDNA文库构建及其应用［D］.武汉:华中农业大学, 2007.

［22］GUAN G Q, MOREAU E, BRISSEAU N, et al. Determination of erythrocyte susceptibility of Chinese sheep (Tan mutton breed) and French sheep (Vendeen breed) to Babesia sp. BQ1 (Lintan) by in vitro culture［J］. Vet Parasitol, 2010, 170(1-2): 37-43.

［23］GUAN G Q, MOREAU E, LIU J L, et al. BQP35 is a novel member of the intrinsically unstructured protein (IUP) family which is a potential antigen for the sero-diagnosis of Babesia sp. BQ1 (Lintan) infection［J］. Vet Parasitol, 2012, 187(3-4): 421-430.

［24］GUAN G Q, CHAUVIN A, YIN H, et al. Course of infection by Babesia sp. BQ1 (Lintan) and B. divergens in sheep depends on the production of IFNgamma and IL10［J］. Parasite Immunol, 2010, 32 (2): 143-152.

［25］GULLO C A, TEOH G. Heat shock proteins: to present or not, that is the question［J］. Immunol Lett, 2004, 94(1-2): 1-10.

［26］BUCHANAN K L. Stress and the evolution of condition-dependent signals［J］. Trends Ecol Evol, 2000, 15(4): 156-160.

［27］KUMAR R, MUSIYENKO A, BARIK S. The heat shock protein 90 of Plasmodium falciparum and antimalarial activity of its inhibitor, geldanamycin［J］. Malar J, 2003, 2: 30.

［28］ECHEVERRIA P C, MATRAJT M, HARB O S, et al. Toxoplasma gondii Hsp90 is a potential drug target whose expression and subcellular localization are developmentally regulated［J］. J Mol Biol, 2005, 350(4): 723-734.

［29］BAUM J, RICHARD D, HEALER J, et al. A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites［J］. J Biol Chem, 2006, 281 (8): 5197-5208.

［30］DOBROWOLSKI J M, CARRUTHERS V B, SIBLEY L D. Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii［J］. Mol Microbiol, 1997, 26 (1): 163-173.

［31］GASKINS E, GILK S, DEVORE N, et al. Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii［J］. J Cell Biol, 2004, 165 (3): 383-393.

［32］DUNKER A K, LAWSON J D, BROWN C J, et al. Intrinsically disordered protein［J］. J Mol Graph Model, 2001, 19(1): 26-59.

［33］DYSON H J, WRIGHT P E. Intrinsically unstructured proteins and their functions［J］. Nat Rev Mol Cell Biol, 2005, 6(3): 197-208.

［34］JANOO R, MUSOKE A, WELLS C, et al. A Rab1 homologue with a novel isoprenylation signal provides insight into the secretory pathway of Theileria parva［J］. Mol Biochem Parasitol, 1999, 102(1): 131-143.

［35］ZHOU J, MULENGA A, YAMASAKI M, et al. Babesia gibsoni: molecular cloning and characterization of Rab6 and Rab11 homologues［J］. Exp Parasitol, 2002, 101(4): 210-214.

［36］STENMARK H, OLKKONEN V M. The Rab GTPase family［J］. Genome Biol, 2001, 2(5): REVIEWS3007.

［37］TEBELE N, SKILTON R A, KATENDE J, et al. Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina［J］. J Clin Microbiol, 2000, 38(6): 2240-2247.

［38］PETTER M, LEE C C, BYRNE T J, et al. Expression of P. falciparum var genes involves exchange of the histone variant H2A.Z at the promoter［J］. PLoS Pathog, 2011, 7(2): e1001292.

［39］DALMASSO M C, ONYANGO D O, NAGULESWARAN A, et al. Toxoplasma H2A variants reveal novel insights into nucleosome composition and functions for this histone family［J］. J Mol Biol, 2009, 392(1): 33-47.

［40］GARDNER M J, BISHOP R, SHAH T, et al. Genome sequence of Theileria parva, a bovine pathogen that transforms lymphocytes［J］. Science, 2005, 309 (5731): 134-137.

［41］BRAYTON K A, LAU A O, HERNDON D R, et al. Genome sequence of Babesia bovis and comparative analysis of apicomplexan hemoprotozoa［J］. PLoS Pathog, 2007, 3(10): 1401-1413.

［42］PAIN A, RENAULD H, BERRIMAN M, et al. Genome of the host-cell transforming parasite Theileria annulata compared with T. parva［J］. Science, 2005, 309(5731): 131-133.

［43］MCLAUGHLIN K J, JENKINS J L, KIELKOPF C L. Large favorable enthalpy changes drive specific RNA recognition by RNA recognition motif proteins［J］. Biochemistry, 2011, 50(9): 1429-1431.

［44］MARIS C, DOMINGUEZ C, ALLAIN F H. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression［J］. FEBS J, 2005, 272(9): 2118-2131.

［45］CLERY A, BLATTER M, ALLAIN F H. RNA recognition motifs: boring? Not quite［J］. Curr Opin Struct Biol, 2008, 18(3): 290-298.

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