牛环形泰勒虫诊断性靶基因片段的筛选及鉴定
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摘要
通过对牛环形泰勒虫Tams1基因的不同片段进行克隆表达,以期得出该基因疏水区对蛋白表达的影响及筛选的目的蛋白作为ELISA抗原的特异性。利用3对不同的特异性引物对Tams1基因的三段区域(全长基因、无N端及C端疏水区、仅缺乏N端的疏水区)进行克隆,最终构建三种重组表达载体。经电泳分析筛选获得可溶性表达重组质粒,目的蛋白经亲和层析纯化后进行Western-blot分析。结果显示,在37℃条件下,经终浓度为1mmol/L IPTG诱导表达4h后,无疏水区的靶重组质粒表达出53ku的可溶性蛋白,其表达量达1.39mg/mL;而含全长基因或C端疏水区的质粒分别表达55ku、56ku的包涵体蛋白。Western-blot分析结果表明,筛选得到的可溶性蛋白能被环形泰勒虫阳性血清特异性识别,具有良好的反应原性。结果表明,该蛋白可作为潜在的进行牛环形泰勒虫病流行病学调查的候选抗原。
Effects of Tams1gene hydrophobic domains on its expression and specificity of screened protein used as ELISA antigen were studied by cloning and expressing the different gene fragments.Three fragments including the complete gene,the complete gene lacking N-terminal and C-terminal hydrophobic domain,and the gene fragment without the sequences coding for N-terminal hydrophobic domains of Tams1gene were amplified by PCR using three sets of specific primers.Then three recombinant plasmids were constructed and finally 3high soluble expression recombinant plasmids were screened and the expressed protein was analyzed by SDS-PAGE and Western-blot.In result,the recombinant plasmid without the sequences encoding for both hydrophobic domains could express 53ku soluble protein when induced with 1mmol/L IPTG for 4hat 37℃,and its expression level reached 1.39mg/mL.Soluble expression content of the recombinant plasmid with the complete gene or C-terminal hydrophobic domain were few and were mainly 55ku and 56ku proteins in a form of inclusion bodies,respectively.Western-blot analysis showed that the soluble protein could be specifically recognized by the positive serum against Theileria annulata,so it had good reactionogenicity.The result showed that acquired soluble protein could become a useful tool to survey the epidemiology of Theileria annulata.
Effects of Tams1gene hydrophobic domains on its expression and specificity of screened protein used as ELISA antigen were studied by cloning and expressing the different gene fragments.Three fragments including the complete gene,the complete gene lacking N-terminal and C-terminal hydrophobic domain,and the gene fragment without the sequences coding for N-terminal hydrophobic domains of Tams1gene were amplified by PCR using three sets of specific primers.Then three recombinant plasmids were constructed and finally 3high soluble expression recombinant plasmids were screened and the expressed protein was analyzed by SDS-PAGE and Western-blot.In result,the recombinant plasmid without the sequences encoding for both hydrophobic domains could express 53ku soluble protein when induced with 1mmol/L IPTG for 4hat 37℃,and its expression level reached 1.39mg/mL.Soluble expression content of the recombinant plasmid with the complete gene or C-terminal hydrophobic domain were few and were mainly 55ku and 56ku proteins in a form of inclusion bodies,respectively.Western-blot analysis showed that the soluble protein could be specifically recognized by the positive serum against Theileria annulata,so it had good reactionogenicity.The result showed that acquired soluble protein could become a useful tool to survey the epidemiology of Theileria annulata.
引文
[1]SCHNITTGER L,KATZER F,BIERMANN R,et al.Characterization of a polymorphic Theileria annulata surface protein(TaSP)closely related to PIM of Theileria parva:Implications for use in diagnostic tests and subunit vaccines[J].Mol Biochem Parasit,2002,120(2):247-256.
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[6]简子健,黄家雨,马素贞,等.牛环形泰勒虫巢式PCR诊断方法的建立[J].新疆农业大学学报,2008,31(6):59-62.JIAN Zi-jian,HUANG Jia-yu,MA Su-zhen,et al.Development of nested PCR for detection of bovine Theileria annulata in Xinjiang[J].Journal of Xinjiang Agricultural University,2008,31(6):59-62.(in Chinese)
[7]王振宝,刘启生,哈森,等.牛环形泰勒虫病二温式PCR诊断方法的建立及初步应用[J].动物医学进展,2012,33(5):59-63.WANG Zhen-bao,LIU Qi-sheng,HA Sen,et al.Establishment and primary application of a two-temperature PCR method for detection Theileria annulatainfection in cattle[J].Progress in Veterinary Medicine,2012,33(5):59-63.(in Chinese)
[8]GUBBELS M J,D'OLIVEIRA C,JONGEJAN F.Development of an indirect Tams1enzyme-linked immunosorbent assay for diagnosis of Theileria annulatainfection in cattle[J].Clin Diagn Lab Immunol,2000,7(3):404-411.
[9]黄家雨.新疆牛环形泰勒虫Tams1基因的克隆、表达与巢式PCR诊断方法的建立[D].乌鲁木齐:新疆农业大学,2008.HUANG Jia-yu.The cloning and expression of the merozoite/piroplasm surface antigen of the bovine Theileria annulata in Xinjiang and establishment of nested PCR on Theileria annulata[D].Urumqi:Xinjiang Agricultural University,2008.(in Chinese)
[10]GLASCODINE J,TETLEY L,TAIT A,et al.Developmental expression of a Theileria annulata merozoite surface antigen[J].Mol Biochem Parasitol,1990,40(1):105-112.
[11]KATZER F,MCKELLAR S,BEN MILED L,et al.Selection for antigenic diversity of Tams1,the major merozoite antigen of Theileria annulata[J].Ann N Y Acad Sci,1998,849:96-108.
[12]OLIVEIRA C,TIJHAAR E J,SHIELS B R,et al.Expression of genes encoding two major Theileria annulata merozoite surface antigens in Escherichia coli and a Salmonella typhimuriumaroA vaccine strain[J].Gene,1996,172(1):33-39.
[13]沙塔尔·卡哈尔,曹雯丽,张扬,等.吐鲁番部分疫区牛环形泰勒虫病流行病学调查[J].新疆农业科学,2013,50(6):1161-1164.SHATAER Kahaer,CAO Wen-li,ZHANG Yang,et al.Epidemiological investigation of infection of Theileria annulata for cattle in some epidemic areas of Turpan[J].Xinjiang Agricultural Sciences,2013,50(6):1161-1164.(in Chinese)
[14]吴永红,刘玲,王真,等.南疆部分散养户牛场梨形虫及其媒介蜱感染情况的调查[J].新疆农业科学,2011,48(10):1918-1922.WU Yong-hong,LIU Ling,WANG Zhen,et al.Investigation of vector ticks and piroplasmosis infection in scattered feeding cattle farm in Southern Xinjiang[J].Xinjiang Agricultural Sciences,2011,48(10):1918-1922.(in Chinese)
[15]RAJENDRAN C,RAY DBANSAL G.Expression of gene encoding immunodominant merozoite surface protein of Theileria annulata in Escherichia coli[J].Indian J Biotechnol,2008,7(2):200-203.
[2]宋世荣,李良荣,史晓,等.我国北方各省(区)牛环形泰勒焦虫病调查报告[J].贵州畜牧兽医,2000,24(3):9-10.SONG Shi-rong,LI Liang-rong,SHI Xiao,et al.Investigation of infection of Theileria annulata for cattle in northern provinces of China[J].Guizhou Animal Science and Veterinary Medicine,2000,24(3):9-10.(in Chinese)
[3]D’OLIVEIRA C,VAN DER WEIDE M,HABELA M A,et al.Detection of Theileria annulatain blood samples of carrier cattle by PCR[J].J Clin Microbiol,1995,33(10):2665-2669.
[4]AZIZI H,SHIRAN B,FARZANEH DEHKORDI A,et al.Detection of Theileria annulata by PCR and its comparison with smear method in native carrier cows[J].Biotechnol,2008,7(3):574-577.
[5]DUMANLI N,AKTAS M,CETINKAYA B,et al.Prevalence and distribution of tropical theileriosis in eastern Turkey[J].Vet Parasitol,2005,127(1):9-15.
[6]简子健,黄家雨,马素贞,等.牛环形泰勒虫巢式PCR诊断方法的建立[J].新疆农业大学学报,2008,31(6):59-62.JIAN Zi-jian,HUANG Jia-yu,MA Su-zhen,et al.Development of nested PCR for detection of bovine Theileria annulata in Xinjiang[J].Journal of Xinjiang Agricultural University,2008,31(6):59-62.(in Chinese)
[7]王振宝,刘启生,哈森,等.牛环形泰勒虫病二温式PCR诊断方法的建立及初步应用[J].动物医学进展,2012,33(5):59-63.WANG Zhen-bao,LIU Qi-sheng,HA Sen,et al.Establishment and primary application of a two-temperature PCR method for detection Theileria annulatainfection in cattle[J].Progress in Veterinary Medicine,2012,33(5):59-63.(in Chinese)
[8]GUBBELS M J,D'OLIVEIRA C,JONGEJAN F.Development of an indirect Tams1enzyme-linked immunosorbent assay for diagnosis of Theileria annulatainfection in cattle[J].Clin Diagn Lab Immunol,2000,7(3):404-411.
[9]黄家雨.新疆牛环形泰勒虫Tams1基因的克隆、表达与巢式PCR诊断方法的建立[D].乌鲁木齐:新疆农业大学,2008.HUANG Jia-yu.The cloning and expression of the merozoite/piroplasm surface antigen of the bovine Theileria annulata in Xinjiang and establishment of nested PCR on Theileria annulata[D].Urumqi:Xinjiang Agricultural University,2008.(in Chinese)
[10]GLASCODINE J,TETLEY L,TAIT A,et al.Developmental expression of a Theileria annulata merozoite surface antigen[J].Mol Biochem Parasitol,1990,40(1):105-112.
[11]KATZER F,MCKELLAR S,BEN MILED L,et al.Selection for antigenic diversity of Tams1,the major merozoite antigen of Theileria annulata[J].Ann N Y Acad Sci,1998,849:96-108.
[12]OLIVEIRA C,TIJHAAR E J,SHIELS B R,et al.Expression of genes encoding two major Theileria annulata merozoite surface antigens in Escherichia coli and a Salmonella typhimuriumaroA vaccine strain[J].Gene,1996,172(1):33-39.
[13]沙塔尔·卡哈尔,曹雯丽,张扬,等.吐鲁番部分疫区牛环形泰勒虫病流行病学调查[J].新疆农业科学,2013,50(6):1161-1164.SHATAER Kahaer,CAO Wen-li,ZHANG Yang,et al.Epidemiological investigation of infection of Theileria annulata for cattle in some epidemic areas of Turpan[J].Xinjiang Agricultural Sciences,2013,50(6):1161-1164.(in Chinese)
[14]吴永红,刘玲,王真,等.南疆部分散养户牛场梨形虫及其媒介蜱感染情况的调查[J].新疆农业科学,2011,48(10):1918-1922.WU Yong-hong,LIU Ling,WANG Zhen,et al.Investigation of vector ticks and piroplasmosis infection in scattered feeding cattle farm in Southern Xinjiang[J].Xinjiang Agricultural Sciences,2011,48(10):1918-1922.(in Chinese)
[15]RAJENDRAN C,RAY DBANSAL G.Expression of gene encoding immunodominant merozoite surface protein of Theileria annulata in Escherichia coli[J].Indian J Biotechnol,2008,7(2):200-203.