日本血吸虫SjIrV1的基因特性及免疫保护效果
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摘要
钙结合蛋白是日本血吸虫生长发育不可缺少的蛋白,具有非常广泛而重要的功能。在课题组日本血吸虫体被表膜蛋白研究基础上,利用PCR技术克隆了中国大陆株日本血吸虫66 kDa钙结合蛋白(SjIrV1)编码基因的cDNA序列,BLAST分析与菲律宾株日本血吸虫SjIrV1 cDNA编码序列一致,荧光定量PCR分析表明该基因在童虫和成虫期不同发育阶段均有表达,其中在35 d和42 d成虫中表达量较高,在42 d雌虫中该基因表达水平远高于42 d雄虫。构建重组表达质粒pET28a(+)-SjIrV1,在大肠杆菌中成功诱导表达,重组蛋白主要以可溶性形式存在,通过高效液相色谱法(RP-HPLC)以及串联质谱法(MS/MS)鉴定所获蛋白为目的蛋白SjIrV1。蛋白质印迹(Western blotting)分析结果显示重组蛋白能被感染日本血吸虫鼠血清和免疫鼠血清所识别,SjIrV1蛋白在虫体各发育阶段中均表达。免疫荧光染色实验观察表明SjIrV1主要分布在日本血吸虫成虫的表膜。应用重组蛋白免疫BALB/c小鼠后,免疫鼠血清中检测到较高水平的特异性IgG、IgG1和IgG2a抗体。结果表明SjIrV1可能在日本血吸虫的生长发育过程中起着重要作用。
Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum.Based on our primary study on tegument surface proteins of S.japonicun,a cDNA encoding a 66 kDa calcium-binding protein of S.japonicum(Chinese strain) was cloned,sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S.japonicum.The expression of SjIrV1 were detected by Real-time PCR,using cDNA templates isolated from 7,14,21,28,35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages,and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms.The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a(+) vector and transformed into competent Escherichia coli BL21 for expression.The recombinant protein was purified using a Ni-NTA purification system,and confirmed by high performance liquid chromatography(RP-HPLC) and tandem mass spectrometry(MS/MS).Western blotting analysis showed that recombinant SjIrV1(rSjIrV1) could be recognized by the S.japonicum infected mouse serum and the mouse serum specific to rSjIrV1,respectively.Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms.ELISA test revealed that IgG,IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice.The study suggested that rSjIrV1 might play an important role in the development of S.japonicum.
Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum.Based on our primary study on tegument surface proteins of S.japonicun,a cDNA encoding a 66 kDa calcium-binding protein of S.japonicum(Chinese strain) was cloned,sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S.japonicum.The expression of SjIrV1 were detected by Real-time PCR,using cDNA templates isolated from 7,14,21,28,35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages,and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms.The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a(+) vector and transformed into competent Escherichia coli BL21 for expression.The recombinant protein was purified using a Ni-NTA purification system,and confirmed by high performance liquid chromatography(RP-HPLC) and tandem mass spectrometry(MS/MS).Western blotting analysis showed that recombinant SjIrV1(rSjIrV1) could be recognized by the S.japonicum infected mouse serum and the mouse serum specific to rSjIrV1,respectively.Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms.ELISA test revealed that IgG,IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice.The study suggested that rSjIrV1 might play an important role in the development of S.japonicum.
引文
[1]Schistosomiasis Fact Sheet[EB/OL].[2012-12-10].http://www.who.int/mediacentre/factsheets/fs115/e n/index.html.
[2]Schistosomiasis:Centers for Disease Control and Prevention[EB/OL].[2012-12-10].http://www.cdc.gov/parasites/schistosomiasis/.
[3]Lei ZL,Zheng H,Zhang LJ,et al.Schistosomiasis status in People’s Republic of China in2010.Chin J Schistosom Control,2011,23(6):599604(in Chinese).雷正龙,郑浩,张利娟,等.2010年全国血吸虫病疫情通报.中国血吸虫病防治杂志,2011,23(6):599604.
[4]Redam CA,Robertson A,Fallon PG,et al.Praziquantel:an urgent and exciting challenge.Parasitol Today,1996,12(1):1420.
[5]Gryseels B,Polderman AM.Morbidity,due to Schistosomiasis mansoni,and its control in Subsaharan Africa.Parasitol Today,1991,7(9):244248.
[6]Taylor DW.Schistosome vaccines.Cell Mol Life Sci,1991,47(2):152157.
[7]Liang YS,Li HJ,Dai JR,et al.Studies on resistance of schistosoma to praziquantel XIII resistance of Schistosoma japonicum to praziquantel is experimentally induced in laboratory.Chin J Schistosom Control,2011,23(06):605610(in Chinese).梁幼生,李洪军,戴建荣,等.血吸虫对吡喹酮抗药性的研究ⅩⅢ日本血吸虫吡喹酮抗药性的实验诱导.中国血吸虫病防治杂志,2011,23(06):605610.
[8]Bergquist NR,Leonardo LR,Mitchell GF.Vaccine-linked chemotherapy:can schistosomiasis control benefit from an integrated approach?Trends Parasitol,2005,21(3):112117.
[9]Thomas RH,Timothy DT,Mette S.Molecular cloning and expression of SmIrV1,a Schistosoma mansoni antigen with similarity to calnexin,calreticulin and OvRal1.J Biol Chem,1993,268(11):76927698.
[10]Hooker CW,Brindley PJ.Cloning of cDNA encoding SjIrV1,a Schistosoma japonicum calcium-binding protein similar to calnexin,and expression of the recombinant protein in Escherichia coil.Biochimi Biophys Acta,1999,1429(2):331341.
[11]Zhang M,Fu ZQ,Lin JJ.Researches on proteomics of Schistosoma japonicum.Chin J Animal Infect Dis,2011,19(3):6168(in Chinese).张旻,傅志强,林矫矫.日本血吸虫蛋白质组学研究.中国动物传染病学报,2011,19(3):6168.
[12]Santos TM,Johnston DA,Azevedo V,et al.Analysis of the gene expression profile of Schistosoma mansoni cercariae using the expressed sequence tag approach.Mol Biochem Parasitol,1999,103(1):7997.
[13]Stewart GL,Kim I,Shupe K,et al.Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite.Parasitology,1992,78(5):849855.
[14]Stewart TJ,Smith AL,Havercroft JC.Analysis of the complete sequence of a muscle calcium-binding protein of Schistosoma mansoni.Parasitology,1992,105(3):399408.
[15]Al-Sherbiny M,Osman A,Barakat R,et al.In vitro cellular and humoral responses to Schistosoma mansoni vaccine candidate antigens.Acta Trop,2003,88(2):11730.
[16]Bergeron JJ,Brenner MB,Thomas DY,et al.Calnexin:a membrane-bound chaperone of the endoplasmic reticulum.Trends Biochem Sci,1994,19(3):124128.
[17]Rzepecka J,Rausch S,Klotz C,et al.Calreticulinfrom the intestinal nematode Heligmosomoides polygyrus is a Th2-skewing protein and interacts with murine scavenger receptor-A.Mol Immunol,2009,46(6):11091119.
[18]Watanabe D,Yamada K,Nishina Y,et al.Molecular cloning of a novel Ca 2+-binding protein(calmegin)specifically expressed during male meiotic germ cell development.J Biol Chem,1994,269(10):77447749.
[19]Unnasch TR,Gallin MY,Soboslay PT,et al.Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae.J Clin Invest,1988,82(1):262–269.
[20]Tang WH,Zhang JL,Wang ZY,et al.The cause of deviation made in determining the molecular weight of His-tag fusion proteins by SDS-PAGE.J Plant Physiol Mol Biol,2000,26(1):6468(in Chinese).唐威华,张景六,王宗阳,等.SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因.植物生理学报,2000,26(1):6468.
[21]He YX,Yang HZ.Physiological studies on the post-cercarial development of Schistosoma japoncum.Curr Zool,1980,26(01):3241(in Chinese).何毅勋,杨惠中.日本血吸虫发育的生理学研究.动物学报,1980,26(01):3241.
[22]Balakier H,Dziak E,Sojecki A,et al.Calcium-binding proteins and calcium-release channels in human maturing oocytes,pronuclear zygotes and early preimplantation embryos.Hum Reprod,2002,17(11):29382947.
[23]Bergeron JJ,Brenner MB,Thomas DY,et al.Calnexin:a membrane-bound chaperone of the endoplasmic reticulum.Trends Biochem Sci,1994,19(3):124128.
[24]Bergquist R,Utzinger J,McManus DP.Trick or treat:the role of vaccines in integrated schistosomiasis control.PLoS Neglect Trop Dis,2008,2(6):e244.
[25]Finkelman FD,Holmes J,Katona IM,et al.Lymphokine control of in vivo immunoglobulin isotype selection.Annu Rev Immunol,1990,8:303333.
[26]Golding B,Scott DE.Vaccine strategies:targeting helper T cell responses.Ann N Y Acad Sci,1995,754:126137.
[27]Xu XD,Zhang DM,Sun W,et al.A Schistosoma japonicum chimeric protein with a novel adjuvant induced a polarized Th1immune response and protection against liver egg burdens.BMC Infect Dis,2009,9:54.
[28]Zhang L,Chen JP.Advances in the study of candidate vaccines of Schistosoma japonicum.Paras Infect Dis,2005,3(01):3134(in Chinese).张莉,陈建平.日本血吸虫疫苗候选分子的研究进展.寄生虫病与感染性疾病,2005,3(01):3134.
[2]Schistosomiasis:Centers for Disease Control and Prevention[EB/OL].[2012-12-10].http://www.cdc.gov/parasites/schistosomiasis/.
[3]Lei ZL,Zheng H,Zhang LJ,et al.Schistosomiasis status in People’s Republic of China in2010.Chin J Schistosom Control,2011,23(6):599604(in Chinese).雷正龙,郑浩,张利娟,等.2010年全国血吸虫病疫情通报.中国血吸虫病防治杂志,2011,23(6):599604.
[4]Redam CA,Robertson A,Fallon PG,et al.Praziquantel:an urgent and exciting challenge.Parasitol Today,1996,12(1):1420.
[5]Gryseels B,Polderman AM.Morbidity,due to Schistosomiasis mansoni,and its control in Subsaharan Africa.Parasitol Today,1991,7(9):244248.
[6]Taylor DW.Schistosome vaccines.Cell Mol Life Sci,1991,47(2):152157.
[7]Liang YS,Li HJ,Dai JR,et al.Studies on resistance of schistosoma to praziquantel XIII resistance of Schistosoma japonicum to praziquantel is experimentally induced in laboratory.Chin J Schistosom Control,2011,23(06):605610(in Chinese).梁幼生,李洪军,戴建荣,等.血吸虫对吡喹酮抗药性的研究ⅩⅢ日本血吸虫吡喹酮抗药性的实验诱导.中国血吸虫病防治杂志,2011,23(06):605610.
[8]Bergquist NR,Leonardo LR,Mitchell GF.Vaccine-linked chemotherapy:can schistosomiasis control benefit from an integrated approach?Trends Parasitol,2005,21(3):112117.
[9]Thomas RH,Timothy DT,Mette S.Molecular cloning and expression of SmIrV1,a Schistosoma mansoni antigen with similarity to calnexin,calreticulin and OvRal1.J Biol Chem,1993,268(11):76927698.
[10]Hooker CW,Brindley PJ.Cloning of cDNA encoding SjIrV1,a Schistosoma japonicum calcium-binding protein similar to calnexin,and expression of the recombinant protein in Escherichia coil.Biochimi Biophys Acta,1999,1429(2):331341.
[11]Zhang M,Fu ZQ,Lin JJ.Researches on proteomics of Schistosoma japonicum.Chin J Animal Infect Dis,2011,19(3):6168(in Chinese).张旻,傅志强,林矫矫.日本血吸虫蛋白质组学研究.中国动物传染病学报,2011,19(3):6168.
[12]Santos TM,Johnston DA,Azevedo V,et al.Analysis of the gene expression profile of Schistosoma mansoni cercariae using the expressed sequence tag approach.Mol Biochem Parasitol,1999,103(1):7997.
[13]Stewart GL,Kim I,Shupe K,et al.Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite.Parasitology,1992,78(5):849855.
[14]Stewart TJ,Smith AL,Havercroft JC.Analysis of the complete sequence of a muscle calcium-binding protein of Schistosoma mansoni.Parasitology,1992,105(3):399408.
[15]Al-Sherbiny M,Osman A,Barakat R,et al.In vitro cellular and humoral responses to Schistosoma mansoni vaccine candidate antigens.Acta Trop,2003,88(2):11730.
[16]Bergeron JJ,Brenner MB,Thomas DY,et al.Calnexin:a membrane-bound chaperone of the endoplasmic reticulum.Trends Biochem Sci,1994,19(3):124128.
[17]Rzepecka J,Rausch S,Klotz C,et al.Calreticulinfrom the intestinal nematode Heligmosomoides polygyrus is a Th2-skewing protein and interacts with murine scavenger receptor-A.Mol Immunol,2009,46(6):11091119.
[18]Watanabe D,Yamada K,Nishina Y,et al.Molecular cloning of a novel Ca 2+-binding protein(calmegin)specifically expressed during male meiotic germ cell development.J Biol Chem,1994,269(10):77447749.
[19]Unnasch TR,Gallin MY,Soboslay PT,et al.Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae.J Clin Invest,1988,82(1):262–269.
[20]Tang WH,Zhang JL,Wang ZY,et al.The cause of deviation made in determining the molecular weight of His-tag fusion proteins by SDS-PAGE.J Plant Physiol Mol Biol,2000,26(1):6468(in Chinese).唐威华,张景六,王宗阳,等.SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因.植物生理学报,2000,26(1):6468.
[21]He YX,Yang HZ.Physiological studies on the post-cercarial development of Schistosoma japoncum.Curr Zool,1980,26(01):3241(in Chinese).何毅勋,杨惠中.日本血吸虫发育的生理学研究.动物学报,1980,26(01):3241.
[22]Balakier H,Dziak E,Sojecki A,et al.Calcium-binding proteins and calcium-release channels in human maturing oocytes,pronuclear zygotes and early preimplantation embryos.Hum Reprod,2002,17(11):29382947.
[23]Bergeron JJ,Brenner MB,Thomas DY,et al.Calnexin:a membrane-bound chaperone of the endoplasmic reticulum.Trends Biochem Sci,1994,19(3):124128.
[24]Bergquist R,Utzinger J,McManus DP.Trick or treat:the role of vaccines in integrated schistosomiasis control.PLoS Neglect Trop Dis,2008,2(6):e244.
[25]Finkelman FD,Holmes J,Katona IM,et al.Lymphokine control of in vivo immunoglobulin isotype selection.Annu Rev Immunol,1990,8:303333.
[26]Golding B,Scott DE.Vaccine strategies:targeting helper T cell responses.Ann N Y Acad Sci,1995,754:126137.
[27]Xu XD,Zhang DM,Sun W,et al.A Schistosoma japonicum chimeric protein with a novel adjuvant induced a polarized Th1immune response and protection against liver egg burdens.BMC Infect Dis,2009,9:54.
[28]Zhang L,Chen JP.Advances in the study of candidate vaccines of Schistosoma japonicum.Paras Infect Dis,2005,3(01):3134(in Chinese).张莉,陈建平.日本血吸虫疫苗候选分子的研究进展.寄生虫病与感染性疾病,2005,3(01):3134.