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
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摘要
柔嫩艾美耳球虫是一种重要的寄生于鸡盲肠细胞内的寄生原虫,可引起危害严重的鸡球虫病。目前,对鸡球虫病的预防和治疗主要依靠抗球虫药,而药物的广泛使用使球虫产生了严重的耐药性。为研究球虫耐药性产生的机制,本实验室前期对诱导的地克珠利耐药株、马杜拉霉素耐药株和亲本敏感株进行转录组测序,经分析发现TCP-1亚基α为耐药株和敏感株的差异表达基因且在耐药株上调表达。本文以柔嫩艾美耳球虫敏感株为模板,成功克隆出TCP-1亚基α基因,构建了原核表达重组质粒p ET-Et TCP-1α,并成功诱导表达了重组蛋白r Et TCP-1α,用纯化的重组蛋白免疫新西兰大白兔和BALB/C小鼠获得多克隆抗体。用荧光定量PCR和Western blot方法检测柔嫩艾美耳球虫敏感株、地克珠利耐药株和马杜拉霉素耐药株TCP-1亚基α基因的m RNA转录和翻译水平,同时检测了该基因在球虫不同发育阶段的转录水平和翻译水平。结果显示,Et TCP-1α在2个耐药株的m RNA转录和蛋白翻译水平明显高于敏感株,而且该蛋白在第二代裂殖子的表达最高;间接免疫荧光实验显示该蛋白主要位于子孢子和第二代裂殖子表面,当虫体入侵DF-1细胞进行裂殖生殖后,该蛋白分布于整个虫体,且表达量明显升高。因此,推测Et TCP-1α在柔嫩艾美耳球虫细胞内的生长发育和耐药性的产生过程中发挥了一定的作用。
Eimeria tenella is an important intracellular parasite that invades cecal epithelial cells of chickens, causing serious coccidiosis. At present, prevention and treatment mainly rely on anti-coccidiosis drugs. As a result, the widespread use of drug causes severe resistance to E. tenella. In our previous study, we analysized differentially expressed genes between two drug-resistant strains(diclazurilresistant and maduramicin-resistant strains) and drug-sensitive strain of E. tenella through RNA sequencing. TCP-1 subunit α of E. tenella, one of the differentially expressed genes, was up-regulated in diclazuril-resistant and maduramicin-resistant strains. In this study, E. tenella TCP-1 subunit α(Et TCP-1α) gene was cloned in PCR and ligated into the prokaryotic expression vector p ET-28 a to construct the recombinant plasmid p ET-Et TCP-1α. The recombination protein r Et TCP-1α was expressed after induction with IPTG and then used to immunize New Zealand rabbits and BALB/C mouse to prepare polyclonal antibodies. Quantitative real time PCR and Western blot were applied to analyze the m RNA transcription and translation levels of TCP-1 subunit α in resistant and sensitive strains and test the expression levels of TCP-1 subunit α in four developmental stages of E. tenella. The results revealed that Et TCP-1α was expressed at significantly higher levels in the two drug-resistant strains than drug-sensitive strains and the highest expression occurred in the second generation merozoite stage. The protein Et TCP-1α was mainly located in the surfaces of sporozoites and the second generation merozoites as shown in IFA. After the sporozoites invaded DF-1 cells, Et TCP-1α was uniformly distributed in the immature and mature schizonts and its expression increased signifi cantly. Therefore, it was presumed that Et TCP-1α played a certain role during the growth in the host cells and development of drug resistance of E. tenella.
Eimeria tenella is an important intracellular parasite that invades cecal epithelial cells of chickens, causing serious coccidiosis. At present, prevention and treatment mainly rely on anti-coccidiosis drugs. As a result, the widespread use of drug causes severe resistance to E. tenella. In our previous study, we analysized differentially expressed genes between two drug-resistant strains(diclazurilresistant and maduramicin-resistant strains) and drug-sensitive strain of E. tenella through RNA sequencing. TCP-1 subunit α of E. tenella, one of the differentially expressed genes, was up-regulated in diclazuril-resistant and maduramicin-resistant strains. In this study, E. tenella TCP-1 subunit α(Et TCP-1α) gene was cloned in PCR and ligated into the prokaryotic expression vector p ET-28 a to construct the recombinant plasmid p ET-Et TCP-1α. The recombination protein r Et TCP-1α was expressed after induction with IPTG and then used to immunize New Zealand rabbits and BALB/C mouse to prepare polyclonal antibodies. Quantitative real time PCR and Western blot were applied to analyze the m RNA transcription and translation levels of TCP-1 subunit α in resistant and sensitive strains and test the expression levels of TCP-1 subunit α in four developmental stages of E. tenella. The results revealed that Et TCP-1α was expressed at significantly higher levels in the two drug-resistant strains than drug-sensitive strains and the highest expression occurred in the second generation merozoite stage. The protein Et TCP-1α was mainly located in the surfaces of sporozoites and the second generation merozoites as shown in IFA. After the sporozoites invaded DF-1 cells, Et TCP-1α was uniformly distributed in the immature and mature schizonts and its expression increased signifi cantly. Therefore, it was presumed that Et TCP-1α played a certain role during the growth in the host cells and development of drug resistance of E. tenella.
引文
[1]索勋,李国清.鸡球虫病学[M].北京:中国农业大学出版社,1998.
[2]Shirley M W,Smith A L,Blake D P.Challenges in the successful control of the avian coccidia[J].Vaccine,2007,25(30):5540-5547.
[3]Waletzky E.A field stram of Eimeria tenella resistant to sulfonamides(abstr)[J].J Parasitol,1954,40(2):24.
[4]陈兆国,史天卫,赵其平,等.鸡球虫田间混合种和柔嫩艾美耳球虫单一种的抗药性试验[J].中国兽医科技,2002,32(6):30-32.
[5]王中宝.安徽三个地区柔嫩艾美尔球虫耐药性分析[D].合肥:安徽农业大学,2013.
[6]韩谦,刘群,张荣春.山东省诸城肉鸡球虫的抗药性调查[J].中国兽医学报,1999,19(1):40-42.
[7]李会.空肠弯曲菌酰胺醇类诱导耐药株与敏感株的组学比较研究[D].北京:中国农业大学,2015.
[8]李琳,冯帅,汪莹,等.鼠伤寒沙门菌ATCC13311诱导耐药株的转录组测序和分析[J].中国兽医学报,2015,35(7):1088-1111.
[9]García-Huertas P,Mejía-Jaramillo A M,González L,et al.Transcriptome and functional genomics reveal the participation of adenine phosphoribosyltransferase in Trypanosoma cruzi resistance to benznidazole[J].J Cell Biochem,2017,118(7):1936-1945.
[10]Pulcini S,Staines H M,Lee A H,et al.Mutations in the Plasmodium falciparum chloroquine resistance transporter,Pf CRT,enlarge the parasite's food vacuole and alter drug sensitivities[J].Sci Rep,2015,5:14552.
[11]赵嵘,胡丽玲,孔繁强,等.PXR基因外显子3甲基化与肠癌细胞对5-氟尿嘧啶的耐药性相关[J].中国生物化学与分子生物学报,2013,29(1):63-69.
[12]周蕾,姜妮,周心娜,等.四个半LIM结构域蛋白1对人肝癌细胞紫杉醇耐药影响的研究[J].中国临床药理学杂志,2017,33(12):1135-1138.
[13]孟靖顺.白念珠菌HSP90 C端结构和功能的研究[D].上海:第二军医大学,2017.
[14]胡文霞.骨膜蛋白在非小细胞肺癌中的作用及相关机制的研究[D].石家庄:河北医科大学,2017.
[15]韩红玉,赵其平,陈兆国,等.柔嫩艾美耳球虫地克珠利抗药株和马杜拉霉素抗药株的实验室诱导[J].中国兽医学报,2004,24(2):138-140.
[16]黄兵,赵其平,吴薛忠,等.柔嫩艾美耳球虫纯种的初步确定和致病性研究[J].上海畜牧兽医通讯,1993(5):18-20.
[17]Tomley F.Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites[J].Methods,1997,13(2):171-176.
[18]Shirley M W.Eimeria species and strains of chickens[M]//Coudert P,Eckert J,Braun R,et al.Biotechnology guidelines on techniques in coccidiosis research.Luxembourg City,Luxembourg:The European Commission DGXII,1995:9-10.
[19]Han H Y,Lin J J,Zhao Q P,et al.Identification of differentially expressed genes in early stages of Eimeria tenella by suppression subtractive hybridization and c DNA microarray[J].Parasit,2010,96(1):95-102.
[20]Xie M Q,Gilbert J M,Fuller A L,et al.A new method for purification of Eimeria tenella merozoites[J].Parasitol Res,1990,76(7):566-569.
[21]Jiang L L,Lin J J,Han H Y,et al.Identification and characterization of Eimeria tenella apical membrane antigen-1(AMA1)[J].PLo S One,2012,7(7):e41115.
[22]Z h a i Q,H u a n g B,D o n g H,e t a l.M o l e c u l a r characterization and immune protection of a new conserved hypothetical protein of Eimeria tenella[J].PLo S One,2016,11(6):e0157678.
[23]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].PLo S One,2016,11(12):e0168132.
[24]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method[J].Methods,2001,25(4):402-408.
[25]聂忠清,吴永刚,蒙建洲.分子伴侣的功能和应用[J].生命科学,2006,18(1):84-89.
[26]Shaw P J,Chaotheing S,Kaewprommal P,et al.Plasmodium parasites mount an arrest response to dihydroartemisinin,as revealed by whole transcriptome shotgun sequencing(RNA-seq)and microarray study[J].BMC Genomics,2015,16:830.
[27]Das S,Shah P,Tandon R,et al.Over-expression of cysteine leucine rich protein is related to SAG resistance in clinical isolates of Leishmania donovani[J].PLo S Negl Trop Dis,2015,9(8):e0003992.
[28]Nicole M V,Natalia L L,Tracy Y E L,et al.Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia--an in vivostudy[J].Proteomics,2006,6(5):1681-1694.
[29]Tanic N,Brkic G,Dimitrijevic B,et al.Identification of differentially expressed m RNA transcripts in drugresistant versus parental human melanoma cell lines[J].Anticancer Res,2006,26(3A):2137-2142.
[30]Narayanan A,Pullepu D,Reddy P K,et al.Defects in protein folding machinery affect cell wall integrity and reduce ethanol tolerance in S.cerevisiae[J].Curr Microbiol,2016,73(1):38-45.
[31]Llorca O,Martín-Benito J,Ritco-Vonsovici M,et al.Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations[J].EMBO J,2000,19(22):5971-5979.
[32]Casalou C,Cyrne L,Rosa M R,et al.Microtubule cytoskeleton perturbation induced by taxol and colchicine affects chaperonin containing TCP-1(CCT)subunit gene expression in tetrahymena cells[J].Biochim Biophys Acta,2001,1522(1):9-21.
[33]Himmelspach R,Nick P,Sch?fer E,et al.Developmental and light-dependent changes of the cytosolic chaperonin containing TCP-1(CCT)subunits in maize seedlings,and the localization in coleoptiles[J].Plant J,1997,12(6):1299-1310.
[34]Seixas C,Cruto T,Tavares A,et al.CCTa and CCTd chaperonin subunits are essential and required for cilia assembly and maintenance in Tetrahymena[J].PLo S One,2010,5(5):e10704.
[35]Yoo B C,Fountoulakis M,Dierssen M,et al.Expression patterns of chaperone proteins in cerebral cortex of the fetus with down syndrome:dysregulation of T-complex protein 1[J].J Neural Transm Suppl,2001,(61):321-334.
[36]Zhang J,Ye C,Ruan X,et al.The chaperonin CCTαis required for efficient transcription and replication of rabies virus[J].Microbiol Immunol,2014,58(10):590-599.
[37]FislováT,Thomas B,Graef K M,et al.Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT[J].J Virol,2010,84(17):8691-8699.
[38]Satish L,Lo N,Gallo P H,et al.Chaperonin containing T-complex polypeptide(CCT)subunit expression in oral mucosal wounds and fibroblasts[J].Cell Stress Chaperones,2011,16(6):675-680.
[39]Satish L,Abdulally A,Oswald D,et al.Differential expression of chaperonin containing T-complex polypeptide(CCT)subunits during fetal and adult skin wound healing[J].Cell Stress Chaperones,2008,13(4):527-533.
[40]Koulikovska M,Podskochy A,Fagerholm P.The expression pattern of the subunit of chaperonin containing T-complex polypeptide 1 and its substrate,alpha-smooth muscle actin,during corneal wound healing[J].Acta Ophthalmol Scand,2005,83(5):543-548.
[41]Yokota S,Yanagi H,Yura T,et al.Cytosolic chaperonincontaining t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycle[J].Eur J Biochem,2001,268(17):4664-4673.
[42]Del Cacho E,Gallego M,Lillehoj HS,et al.IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis[J].Vet Res,2014,45:25.
[43]Haritova A M,Stanilova S A.Enhanced expression of IL-10 in contrast to IL-12B m RNA in poultry with experimental coccidiosis[J].Exp Parasitol,2012,132(3):378-382.
[2]Shirley M W,Smith A L,Blake D P.Challenges in the successful control of the avian coccidia[J].Vaccine,2007,25(30):5540-5547.
[3]Waletzky E.A field stram of Eimeria tenella resistant to sulfonamides(abstr)[J].J Parasitol,1954,40(2):24.
[4]陈兆国,史天卫,赵其平,等.鸡球虫田间混合种和柔嫩艾美耳球虫单一种的抗药性试验[J].中国兽医科技,2002,32(6):30-32.
[5]王中宝.安徽三个地区柔嫩艾美尔球虫耐药性分析[D].合肥:安徽农业大学,2013.
[6]韩谦,刘群,张荣春.山东省诸城肉鸡球虫的抗药性调查[J].中国兽医学报,1999,19(1):40-42.
[7]李会.空肠弯曲菌酰胺醇类诱导耐药株与敏感株的组学比较研究[D].北京:中国农业大学,2015.
[8]李琳,冯帅,汪莹,等.鼠伤寒沙门菌ATCC13311诱导耐药株的转录组测序和分析[J].中国兽医学报,2015,35(7):1088-1111.
[9]García-Huertas P,Mejía-Jaramillo A M,González L,et al.Transcriptome and functional genomics reveal the participation of adenine phosphoribosyltransferase in Trypanosoma cruzi resistance to benznidazole[J].J Cell Biochem,2017,118(7):1936-1945.
[10]Pulcini S,Staines H M,Lee A H,et al.Mutations in the Plasmodium falciparum chloroquine resistance transporter,Pf CRT,enlarge the parasite's food vacuole and alter drug sensitivities[J].Sci Rep,2015,5:14552.
[11]赵嵘,胡丽玲,孔繁强,等.PXR基因外显子3甲基化与肠癌细胞对5-氟尿嘧啶的耐药性相关[J].中国生物化学与分子生物学报,2013,29(1):63-69.
[12]周蕾,姜妮,周心娜,等.四个半LIM结构域蛋白1对人肝癌细胞紫杉醇耐药影响的研究[J].中国临床药理学杂志,2017,33(12):1135-1138.
[13]孟靖顺.白念珠菌HSP90 C端结构和功能的研究[D].上海:第二军医大学,2017.
[14]胡文霞.骨膜蛋白在非小细胞肺癌中的作用及相关机制的研究[D].石家庄:河北医科大学,2017.
[15]韩红玉,赵其平,陈兆国,等.柔嫩艾美耳球虫地克珠利抗药株和马杜拉霉素抗药株的实验室诱导[J].中国兽医学报,2004,24(2):138-140.
[16]黄兵,赵其平,吴薛忠,等.柔嫩艾美耳球虫纯种的初步确定和致病性研究[J].上海畜牧兽医通讯,1993(5):18-20.
[17]Tomley F.Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites[J].Methods,1997,13(2):171-176.
[18]Shirley M W.Eimeria species and strains of chickens[M]//Coudert P,Eckert J,Braun R,et al.Biotechnology guidelines on techniques in coccidiosis research.Luxembourg City,Luxembourg:The European Commission DGXII,1995:9-10.
[19]Han H Y,Lin J J,Zhao Q P,et al.Identification of differentially expressed genes in early stages of Eimeria tenella by suppression subtractive hybridization and c DNA microarray[J].Parasit,2010,96(1):95-102.
[20]Xie M Q,Gilbert J M,Fuller A L,et al.A new method for purification of Eimeria tenella merozoites[J].Parasitol Res,1990,76(7):566-569.
[21]Jiang L L,Lin J J,Han H Y,et al.Identification and characterization of Eimeria tenella apical membrane antigen-1(AMA1)[J].PLo S One,2012,7(7):e41115.
[22]Z h a i Q,H u a n g B,D o n g H,e t a l.M o l e c u l a r characterization and immune protection of a new conserved hypothetical protein of Eimeria tenella[J].PLo S One,2016,11(6):e0157678.
[23]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].PLo S One,2016,11(12):e0168132.
[24]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method[J].Methods,2001,25(4):402-408.
[25]聂忠清,吴永刚,蒙建洲.分子伴侣的功能和应用[J].生命科学,2006,18(1):84-89.
[26]Shaw P J,Chaotheing S,Kaewprommal P,et al.Plasmodium parasites mount an arrest response to dihydroartemisinin,as revealed by whole transcriptome shotgun sequencing(RNA-seq)and microarray study[J].BMC Genomics,2015,16:830.
[27]Das S,Shah P,Tandon R,et al.Over-expression of cysteine leucine rich protein is related to SAG resistance in clinical isolates of Leishmania donovani[J].PLo S Negl Trop Dis,2015,9(8):e0003992.
[28]Nicole M V,Natalia L L,Tracy Y E L,et al.Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia--an in vivostudy[J].Proteomics,2006,6(5):1681-1694.
[29]Tanic N,Brkic G,Dimitrijevic B,et al.Identification of differentially expressed m RNA transcripts in drugresistant versus parental human melanoma cell lines[J].Anticancer Res,2006,26(3A):2137-2142.
[30]Narayanan A,Pullepu D,Reddy P K,et al.Defects in protein folding machinery affect cell wall integrity and reduce ethanol tolerance in S.cerevisiae[J].Curr Microbiol,2016,73(1):38-45.
[31]Llorca O,Martín-Benito J,Ritco-Vonsovici M,et al.Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations[J].EMBO J,2000,19(22):5971-5979.
[32]Casalou C,Cyrne L,Rosa M R,et al.Microtubule cytoskeleton perturbation induced by taxol and colchicine affects chaperonin containing TCP-1(CCT)subunit gene expression in tetrahymena cells[J].Biochim Biophys Acta,2001,1522(1):9-21.
[33]Himmelspach R,Nick P,Sch?fer E,et al.Developmental and light-dependent changes of the cytosolic chaperonin containing TCP-1(CCT)subunits in maize seedlings,and the localization in coleoptiles[J].Plant J,1997,12(6):1299-1310.
[34]Seixas C,Cruto T,Tavares A,et al.CCTa and CCTd chaperonin subunits are essential and required for cilia assembly and maintenance in Tetrahymena[J].PLo S One,2010,5(5):e10704.
[35]Yoo B C,Fountoulakis M,Dierssen M,et al.Expression patterns of chaperone proteins in cerebral cortex of the fetus with down syndrome:dysregulation of T-complex protein 1[J].J Neural Transm Suppl,2001,(61):321-334.
[36]Zhang J,Ye C,Ruan X,et al.The chaperonin CCTαis required for efficient transcription and replication of rabies virus[J].Microbiol Immunol,2014,58(10):590-599.
[37]FislováT,Thomas B,Graef K M,et al.Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT[J].J Virol,2010,84(17):8691-8699.
[38]Satish L,Lo N,Gallo P H,et al.Chaperonin containing T-complex polypeptide(CCT)subunit expression in oral mucosal wounds and fibroblasts[J].Cell Stress Chaperones,2011,16(6):675-680.
[39]Satish L,Abdulally A,Oswald D,et al.Differential expression of chaperonin containing T-complex polypeptide(CCT)subunits during fetal and adult skin wound healing[J].Cell Stress Chaperones,2008,13(4):527-533.
[40]Koulikovska M,Podskochy A,Fagerholm P.The expression pattern of the subunit of chaperonin containing T-complex polypeptide 1 and its substrate,alpha-smooth muscle actin,during corneal wound healing[J].Acta Ophthalmol Scand,2005,83(5):543-548.
[41]Yokota S,Yanagi H,Yura T,et al.Cytosolic chaperonincontaining t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycle[J].Eur J Biochem,2001,268(17):4664-4673.
[42]Del Cacho E,Gallego M,Lillehoj HS,et al.IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis[J].Vet Res,2014,45:25.
[43]Haritova A M,Stanilova S A.Enhanced expression of IL-10 in contrast to IL-12B m RNA in poultry with experimental coccidiosis[J].Exp Parasitol,2012,132(3):378-382.