基于环形泰勒虫Tams1基因多态性的二温式-PCR检测方法的建立
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摘要
【目的】环形泰勒虫作为牛科动物重要的血液原虫对养牛业产生了重大危害。近年来针对该病原Tams1基因的诊断方法及候选疫苗筛选进行了大量工作。但研究证明该基因多态性对诊断及免疫效果存在影响。基于此,本研究对Tams1基因多态性特征进行了分析,并为该病的诊断建立一种实施有效的检测方法。【方法】参考GenBank中公布的环形泰勒虫Tams1基因的核苷酸序列,设计特异性引物。PCR扩增,获得了846 bp的核苷酸片段。将该基因片段与GenBank中主要的12种已知不同区域虫株的相应氨基酸序列进行比较分析。在保守区设计引物,以Tams1基因标准阳性质粒为模板建立环形泰勒虫病的二温式-PCR检测方法。经优化的方法用于田间样品的检测。【结果】该基因编码281个氨基酸,碱性氨基酸48个,酸性氨基酸42个,疏水性氨基酸100个。系统发生树结果显示,环形泰勒虫甘肃株与安哥拉、土耳其、巴林地方株亲缘关系较近。新疆株与意大利、西班牙地方株关系较近,而与甘肃株亲缘关系相对较远。这一结果通过环形泰勒虫不同国家地方株的氨基酸序列对齐结果可以得到进一步的说明。建立的二温式-PCR检测方法,可检测到0.31 fg.μL-1血液中感染虫体的量。特异性结果表明,仅有环形泰勒虫基因组DNA检测为阳性,其它对照虫种基因组模板均表现为阴性,且与感染牛的其它梨形虫无交叉反应。本实验建立的环形泰勒虫二温式-PCR方法对收集的335份野外样品进行检测,阳性检出率为16.33%,显微镜检测阳性率为2.25%,两者符合率100%。与普通PCR比较,二温式-PCR在敏感性方面没有显著差异,但其特异性更高,且该方法反复升温降温时间消耗短。【结论】环形泰勒虫Tams1基因不同地方株存在明显的差异。其氨基酸序列的多态性特征差异显著。因此该基因作为潜在候选抗原应注重多肽疫苗的设计。二温式-PCR方法具有的良好敏感性和特异性,对环形泰勒虫病的流行可以起到提前检测的效果,提高了疾病的诊断效率。
【Objective】It has been widespread concern for gene polymorphism assays in species classification,pathogen detection and vaccine screening.T.annulata is an important blood protozoa in bovine and causes major hazards.In present study the Tams1 amino acid polymorphism of T.annulata was analyzed and a two-temperature PCR detection method was established for T.annulata.【Method】The specific primers were designed of Tams1 gene of T.annulata.A nucleotide fragment of 846 bp in length was obtained by PCR amplification.The gene amino acid sequence was compared and analyzed with 12 known species in different regions of the isolates in GenBank.And other primers were designed in conserved region of Tams1 gene and the detection method of T.annulata was established by two-temperature PCR.The method was used to detect theileriosis in field.【Result】The fragment encoded 281 amino acids,including 48 basic amino acids,42 acidic amino acids and 100 hydrophobic amino acids.Identity analysis showed that Gansu strain of T.annulata had closest relationship with Ankara(Z48739.1),Turkey(AF214911),Bahrain(AF214794) and had little relationship with Xinjiang strain(No.accession number) while had closest relationship with Italy(AF214862) and Spain(AF214815) strains.The results can be testified by sequences alignment.The two-temperature PCR detection method can detect in 0.31 fg.μL-1 blood infection.Specificity results showed that only T.annulata genome DNA test was positive,other control parasite genome template were shown to be negative,and no cross reaction with other infected bovine Piroplasmorida.The two-temperature PCR method of T.annulata was used to test 335 field samples,the positive rate was 16.33%,and the microscopic detection results of 2.25% coincidence rate was 100%.Compared with common PCR,the method had no significant difference in sensitivity.But,two-temperature PCR was higher than common PCR in specificity,and the method took a short time with repeated heating and cooling.【Conclusion】T.annulata Tams1 genes from different strains exist obvious differences.Its amino acid sequence polymorphism feature is significant.Therefore,when the gene is used as a potential candidate antigen attention should be paid to the design of peptide vaccines.Two-temperature PCR method has good sensitivity and specificity.It has significance for early detection,early prevention of T.annulata.
【Objective】It has been widespread concern for gene polymorphism assays in species classification,pathogen detection and vaccine screening.T.annulata is an important blood protozoa in bovine and causes major hazards.In present study the Tams1 amino acid polymorphism of T.annulata was analyzed and a two-temperature PCR detection method was established for T.annulata.【Method】The specific primers were designed of Tams1 gene of T.annulata.A nucleotide fragment of 846 bp in length was obtained by PCR amplification.The gene amino acid sequence was compared and analyzed with 12 known species in different regions of the isolates in GenBank.And other primers were designed in conserved region of Tams1 gene and the detection method of T.annulata was established by two-temperature PCR.The method was used to detect theileriosis in field.【Result】The fragment encoded 281 amino acids,including 48 basic amino acids,42 acidic amino acids and 100 hydrophobic amino acids.Identity analysis showed that Gansu strain of T.annulata had closest relationship with Ankara(Z48739.1),Turkey(AF214911),Bahrain(AF214794) and had little relationship with Xinjiang strain(No.accession number) while had closest relationship with Italy(AF214862) and Spain(AF214815) strains.The results can be testified by sequences alignment.The two-temperature PCR detection method can detect in 0.31 fg.μL-1 blood infection.Specificity results showed that only T.annulata genome DNA test was positive,other control parasite genome template were shown to be negative,and no cross reaction with other infected bovine Piroplasmorida.The two-temperature PCR method of T.annulata was used to test 335 field samples,the positive rate was 16.33%,and the microscopic detection results of 2.25% coincidence rate was 100%.Compared with common PCR,the method had no significant difference in sensitivity.But,two-temperature PCR was higher than common PCR in specificity,and the method took a short time with repeated heating and cooling.【Conclusion】T.annulata Tams1 genes from different strains exist obvious differences.Its amino acid sequence polymorphism feature is significant.Therefore,when the gene is used as a potential candidate antigen attention should be paid to the design of peptide vaccines.Two-temperature PCR method has good sensitivity and specificity.It has significance for early detection,early prevention of T.annulata.
引文
[1]Sutton A J,Karagenc T,Bakirci S,Sarali H,Pekel G,Medley G F.Modelling the transmission dynamics of Theileria annulata:modelstructure and validation for the Turkish context.Parasitology,2012,139(4):441-453.
[2]Luo J X,Lu W S.Cattle Theileriosis in China.Tropical AnimalHealth and Production,1997,29(S4):4-7.
[3]简子健,黄家雨,马素贞,袁江玲,苏贵成,苗中秋,沈炯玉,张兰江,李晓军.新疆牛环形泰勒虫Tams1基因的克隆与表达.中国预防兽医学报,2008,30(10):784-789.Jian Z J,Huang J Y,Ma S Z,Yuan J L,Su G C,Miao Z Q,Shen J Y,Zhang L J,LI X J.Cloning and expression of Tams1 gene from thebovine Theileria annulata in Xinjiang.Chinese Journal of PreventiveVeterinary Medicine,2008,30(10):784-789.(in Chinese)
[4]De Waal D T.The transovarial transmission of Babesia caballi byHyalomma truncatum.Onderstepoort.Veterinary Research,1990,57:99-100.
[5]董倩,黄国强,叶冰莹,陈由强,陈如凯.二温式-PCR在特异短核酸探针合成中的应用.花生学报,2008,37(3):5-10.Dong Q,Huang G Q,Ye B Y,Chen Y Q,Chen R K.Synthesis ofspecific and short RNA probes with two-temperature PCR.Journal ofPeanut Science,2008,37(3):5-10.(in Chinese)
[6]陈亚利,王卫萍,周枚.双温聚合酶链反应检测EB病毒.金陵医院学报,1994,7(3):237-240.Chen Y L,Wang W P,Zhou M.Detection of Epstein-Barr virus DNAby two-temperature PCR cycles.Bulletin of Jinling Hospital,1994,7(3):237-240.(in Chinese)
[7]Glascodine J,Tetley L,Tait A,Brown CGD,Shiels BR.Developmental expression of a Theileria annulata merozoitesurface antigen.Molecular and Biochemical Parasitology,1990,40:105-112.
[8]Dickson J,Shiels B R.Antigenic diversity of a major merozoitesurface molecule in Theileria annulata.Molecular and BiochemicalParasitology,1993,102:305-311.
[9]Shiels B R,d’Oliveira C,McKellar S,Ben-Miled L,Kawazu S,HideG.Selection of diversity at putative glycosylation sites in theimmunodominant merozoite:piroplasm surface antigen of Theileriaparasites.Molecular and Biochemical Parasitology,1995,72:149-162.
[10]Katzer F,McKellar S,Ben Miled L,d’Oliveira C,Shiels B.Selectionfor antigenic diversity of Tams1;the major merozoite antigen ofTheileria annulata.Annals of the New York Academy of Sciences,1998,849:96-108.
[11]Gubbels M J,d'Oliveira C,Jongejan F.Development of an indirectTams1 enzyme-linked immunosorbent assay for diagnosis of Theileriaannulata infection in cattle.Clinical and Vaccine Immunology,2000,7(3):404-411.
[12]d’Oliveira C,Feenstra A,Vos H,Osterhaus A D M E,Shiels BR,Cornelissen A W C A,Jongejan F.Induction of protectiveimmunity to Theileria annulata using two major merozoite surfaceantigens presented by different delivery systems.Vaccine,1997,15:1796-804.
[13]Altay K,Akta M,Dumanli N.PCR-RFLP analysis of the Tams1 geneof Theileria annulata.Turkiye Parazitology Derg,2007,31(3):173-175.
[14]罗金,刘光远,田占成,谢俊仁,张萍.基于18Sr RNA基因序列的我国马梨形虫分类学地位分析.动物分类学报,2011,36(1):99-103.Luo J,Liu G Y,Tian Z C,Xie J R,Zhang P.Taxonomic statusof equine piroplasmid assays based on 18S rRNA gene sequencingin China.Acta Zootaxonomica Sinica,2011,36(1):99-103.(inChinese)
[15]罗金,刘光远,田占成,谢俊仁,沈辉,田美媛.马泰勒虫EMA1基因的克隆及生物学特性分析.中国兽医科学,2012,42(4):352-356.Luo J,Liu G Y,Tian Z C,Xie J R,Shen H,Tian M Y.Cloning andphylogenetic analysis of EMA1 gene of Theileria equi.ChineseVeterinary Science,2012,42(4):352-356.(in Chinese)
[16]罗金,刘光远,田占成,谢俊仁,张萍,沈辉,党根生.基于Hsp70基因的马梨形虫分类学定位分析.中国农业科学,2011,44(17):3694-3700.Luo J,Liu G Y,Tian Z C,Xie J R,Zhang P,Shen H,Dang G S.Taxonomic status assays of Theileria equi based on Hsp70gene.Scientia Agricultura Sinica,2011,44(17):3694-3700.(inChinese)
[17]Bhoora R,Quan M,Matjila P T,Zweygarth E,Guthrie A J,Collins NE.Sequence heterogeneity in the equi merozoite antigen gene(ema-1)of Theileria equi and development of an ema-1-specific TaqManMGBTM assay for the detection of T.equi.Veterinary Parasitology,2010,172:33-45.
[18]Shiels B R,d'Oliveira C,McKellar S,Ben-Miled L,Kawazu S,HideG.Selection of diversity at putative glycosylation sites in theimmunodominant merozoite piroplasm surface antigen of Theileriaparasites.Molecular and Biochemical Parasitology,1995,72:149-162.
[19]M J Gubbels,F Katzer,Hide G,Jongejan F,Shiels B R.Generation ofa Bahrain pattern of diversity in the major merozoite-piroplasmsurface antigen of Theileria annulata.Molecular and BiochemicalParasitology,2000,110:23-32.
[20]Shiels BR,d’Oliveira C,McKellar S,Ben-Miled L,Kawazu S,HideG.Selection of diversity at putative glycosylation sites in theimmunodominant merozoite:piroplasm surface antigen of Theileriaparasites.Molecular and Biochemical Parasitology 1995,72:149-162.
[21]Katzer F,McKellar S,Ben Miled L,d’Oliveira C,Shiels B.Selectionfor antigenic diversity of Tams1;the major merozoite antigen ofTheileria annulata.Annals of the New York Academy of Sciences,1998,849:96-108.
[2]Luo J X,Lu W S.Cattle Theileriosis in China.Tropical AnimalHealth and Production,1997,29(S4):4-7.
[3]简子健,黄家雨,马素贞,袁江玲,苏贵成,苗中秋,沈炯玉,张兰江,李晓军.新疆牛环形泰勒虫Tams1基因的克隆与表达.中国预防兽医学报,2008,30(10):784-789.Jian Z J,Huang J Y,Ma S Z,Yuan J L,Su G C,Miao Z Q,Shen J Y,Zhang L J,LI X J.Cloning and expression of Tams1 gene from thebovine Theileria annulata in Xinjiang.Chinese Journal of PreventiveVeterinary Medicine,2008,30(10):784-789.(in Chinese)
[4]De Waal D T.The transovarial transmission of Babesia caballi byHyalomma truncatum.Onderstepoort.Veterinary Research,1990,57:99-100.
[5]董倩,黄国强,叶冰莹,陈由强,陈如凯.二温式-PCR在特异短核酸探针合成中的应用.花生学报,2008,37(3):5-10.Dong Q,Huang G Q,Ye B Y,Chen Y Q,Chen R K.Synthesis ofspecific and short RNA probes with two-temperature PCR.Journal ofPeanut Science,2008,37(3):5-10.(in Chinese)
[6]陈亚利,王卫萍,周枚.双温聚合酶链反应检测EB病毒.金陵医院学报,1994,7(3):237-240.Chen Y L,Wang W P,Zhou M.Detection of Epstein-Barr virus DNAby two-temperature PCR cycles.Bulletin of Jinling Hospital,1994,7(3):237-240.(in Chinese)
[7]Glascodine J,Tetley L,Tait A,Brown CGD,Shiels BR.Developmental expression of a Theileria annulata merozoitesurface antigen.Molecular and Biochemical Parasitology,1990,40:105-112.
[8]Dickson J,Shiels B R.Antigenic diversity of a major merozoitesurface molecule in Theileria annulata.Molecular and BiochemicalParasitology,1993,102:305-311.
[9]Shiels B R,d’Oliveira C,McKellar S,Ben-Miled L,Kawazu S,HideG.Selection of diversity at putative glycosylation sites in theimmunodominant merozoite:piroplasm surface antigen of Theileriaparasites.Molecular and Biochemical Parasitology,1995,72:149-162.
[10]Katzer F,McKellar S,Ben Miled L,d’Oliveira C,Shiels B.Selectionfor antigenic diversity of Tams1;the major merozoite antigen ofTheileria annulata.Annals of the New York Academy of Sciences,1998,849:96-108.
[11]Gubbels M J,d'Oliveira C,Jongejan F.Development of an indirectTams1 enzyme-linked immunosorbent assay for diagnosis of Theileriaannulata infection in cattle.Clinical and Vaccine Immunology,2000,7(3):404-411.
[12]d’Oliveira C,Feenstra A,Vos H,Osterhaus A D M E,Shiels BR,Cornelissen A W C A,Jongejan F.Induction of protectiveimmunity to Theileria annulata using two major merozoite surfaceantigens presented by different delivery systems.Vaccine,1997,15:1796-804.
[13]Altay K,Akta M,Dumanli N.PCR-RFLP analysis of the Tams1 geneof Theileria annulata.Turkiye Parazitology Derg,2007,31(3):173-175.
[14]罗金,刘光远,田占成,谢俊仁,张萍.基于18Sr RNA基因序列的我国马梨形虫分类学地位分析.动物分类学报,2011,36(1):99-103.Luo J,Liu G Y,Tian Z C,Xie J R,Zhang P.Taxonomic statusof equine piroplasmid assays based on 18S rRNA gene sequencingin China.Acta Zootaxonomica Sinica,2011,36(1):99-103.(inChinese)
[15]罗金,刘光远,田占成,谢俊仁,沈辉,田美媛.马泰勒虫EMA1基因的克隆及生物学特性分析.中国兽医科学,2012,42(4):352-356.Luo J,Liu G Y,Tian Z C,Xie J R,Shen H,Tian M Y.Cloning andphylogenetic analysis of EMA1 gene of Theileria equi.ChineseVeterinary Science,2012,42(4):352-356.(in Chinese)
[16]罗金,刘光远,田占成,谢俊仁,张萍,沈辉,党根生.基于Hsp70基因的马梨形虫分类学定位分析.中国农业科学,2011,44(17):3694-3700.Luo J,Liu G Y,Tian Z C,Xie J R,Zhang P,Shen H,Dang G S.Taxonomic status assays of Theileria equi based on Hsp70gene.Scientia Agricultura Sinica,2011,44(17):3694-3700.(inChinese)
[17]Bhoora R,Quan M,Matjila P T,Zweygarth E,Guthrie A J,Collins NE.Sequence heterogeneity in the equi merozoite antigen gene(ema-1)of Theileria equi and development of an ema-1-specific TaqManMGBTM assay for the detection of T.equi.Veterinary Parasitology,2010,172:33-45.
[18]Shiels B R,d'Oliveira C,McKellar S,Ben-Miled L,Kawazu S,HideG.Selection of diversity at putative glycosylation sites in theimmunodominant merozoite piroplasm surface antigen of Theileriaparasites.Molecular and Biochemical Parasitology,1995,72:149-162.
[19]M J Gubbels,F Katzer,Hide G,Jongejan F,Shiels B R.Generation ofa Bahrain pattern of diversity in the major merozoite-piroplasmsurface antigen of Theileria annulata.Molecular and BiochemicalParasitology,2000,110:23-32.
[20]Shiels BR,d’Oliveira C,McKellar S,Ben-Miled L,Kawazu S,HideG.Selection of diversity at putative glycosylation sites in theimmunodominant merozoite:piroplasm surface antigen of Theileriaparasites.Molecular and Biochemical Parasitology 1995,72:149-162.
[21]Katzer F,McKellar S,Ben Miled L,d’Oliveira C,Shiels B.Selectionfor antigenic diversity of Tams1;the major merozoite antigen ofTheileria annulata.Annals of the New York Academy of Sciences,1998,849:96-108.