巨型住肉孢子虫烯醇酶基因克隆与表达
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摘要
巨型住肉孢子虫(Sarcocystis gigantea)是羊体内一种常见寄生虫,多寄生于羊横纹肌内形成包囊,导致羊肉大量废弃,给畜牧业带来巨大经济损失。本研究尝试筛选能有效诊断住肉孢子虫感染的抗原。通过免疫印迹及质谱分析筛选到巨型住肉孢子虫候选诊断抗原烯醇酶,利用染色体步移技术扩增两侧翼未知序列并表达重组蛋白。应用生物信息学分析和免疫印迹对该蛋白诊断价值进行评价。结果筛选到的候选蛋白为巨型住肉孢子虫烯醇酶(SgENO),克隆得到1 181 bp的基因并获得重组蛋白rSgENO。对rSgENO应用进行初步评价,发现其与其他顶复亚门原虫的烯醇酶氨基酸相似性均较高(71%～92.1%),且能被弓形虫和新孢子虫阳性血清所识别,具有交叉反应性。本研究克隆并表达了巨型住肉孢子虫烯醇酶,后期评价发现该重组蛋白与新孢子虫和弓形虫有较强的交叉反应性,不能用于羊住肉孢子虫的血清学诊断,但有成为疫苗候选分子的潜力。
Sarcocystis gigantea is a globally ubiquitous pathogen that infects sheep. The formation of cyst in striated muscle induces a largely waste of commercial mutton which causes livestock husbandry losses. In this study, we tried to explore the special antigens for serological diagnosis of sarcocystosis. We screened a series of candidate antigens in S. gigantea through Western blot and mass spectrum identification. Whereafter, Genome Walking technology was applied to acquire two flanking sequences of these antigens. Ultimately, purify recombinant proteins was used to evaluate diagnostic value by bioinformatics analysis and Western blot. Results were as follows: Enolase of S. gigantea was finally supposed to a candidate diagnostic antigen, named SgENO. Subsequently, 1 181 bp expressive sequence of SgENO was cloned, and recombinant protein rSgENO was obtained. Nevertheless, it was found to have high homology with enolase amino acids of other Apicomplexa(71%-92.1%), rSgENO could cross react with Toxoplasma gondii and Neospora caninum positive serum. We cloned and expressed the S. gigantea enolase, and the recombinant protein had a cross reaction with other parasitic protozoa positive serum, which made it an irrelevant antigen for Sarcocystis specific diagnose. However, this protein may be a candidate antigen for vaccines.
Sarcocystis gigantea is a globally ubiquitous pathogen that infects sheep. The formation of cyst in striated muscle induces a largely waste of commercial mutton which causes livestock husbandry losses. In this study, we tried to explore the special antigens for serological diagnosis of sarcocystosis. We screened a series of candidate antigens in S. gigantea through Western blot and mass spectrum identification. Whereafter, Genome Walking technology was applied to acquire two flanking sequences of these antigens. Ultimately, purify recombinant proteins was used to evaluate diagnostic value by bioinformatics analysis and Western blot. Results were as follows: Enolase of S. gigantea was finally supposed to a candidate diagnostic antigen, named SgENO. Subsequently, 1 181 bp expressive sequence of SgENO was cloned, and recombinant protein rSgENO was obtained. Nevertheless, it was found to have high homology with enolase amino acids of other Apicomplexa(71%-92.1%), rSgENO could cross react with Toxoplasma gondii and Neospora caninum positive serum. We cloned and expressed the S. gigantea enolase, and the recombinant protein had a cross reaction with other parasitic protozoa positive serum, which made it an irrelevant antigen for Sarcocystis specific diagnose. However, this protein may be a candidate antigen for vaccines.
引文
[1]COLLINS G H,ATKINSON E,CHARLESTON W AG.Studies on Sarcocystis species III:The macrocystic species of sheep[J].N Z Vet J,1979,27(10):204-206.
[2]董辉,陆瑶瑶,杨玉荣.绵羊和山羊住肉孢子虫流行病学及分型研究进展[J].中国人兽共患病学报,2017,33(9):828-836.DONG H,LU Y Y,YANG Y R.Epidemiology and classification of Sarcocystis in sheep and goat[J].Chinese Journal of Zoonoses,2017,33(9):828-836.(in Chinese)
[3]DUBEY J P,CALERO-BERNAL R,ROSENTHALB M,et al.Sarcocystosis of animals and humans[M].2nd ed.Boca Raton:CRC Press,2016.
[4]DUBEY J P,LEEK R G,FAVER R.Prevalence,transmission,and pathogenicity of Sarcocystis gigantea of sheep[J].J Am Vet Med Assoc,1986,188(2):151-154.
[5]MUNDAY B L,OBENDORF D L.Development and growth of Sarcocystis gigantea in experimentally-infected sheep[J].Vet Parasitol,1984,15(3-4):203-211.
[6]MARTNEZ-NAVALN B,ANASTASIO-GINERB,CANO-FRUCTUOSO M,et al.Short communication.Sarcocystisinfection:a major cause of carcass condemnation in adult sheep in Spain[J].Span J Agric Res,2012,10(2):388-392.
[7]CˇERVA L,GUT J.Indirect haemagglutination reaction with antigen of Sarcocystis gigantea(Railliet,1886)Ashford,1977[J].Folia Parasitol(Praha),1983,30(3):223-228.
[8]TENTER A M.Comparison of Dot-ELISA,ELISAand IFAT for the detection of IgG antibodies to Sarcocystis muris in experimentally infected and immunized mice[J].Vet Parasitol,1988,29(2-3):89-104.
[9]MUNDAY B L.The effect of Sarcocystis ovicanis on growth rate and haematocrit in lambs[J].Vet Parasitol,1979,5(2-3):129-135.
[10]HECKEROTH A R,TENTER A M.Comparison of immunological and molecular methods for the diagnosis of infections with pathogenic Sarcocystis species in sheep[J].Tokai J Exp Clin Med,1998,23(6):293-302.
[11]DA SILVA R C,SU C L,LANGONI H.First identification of Sarcocystis tenella(Railliet,1886)Moulé,1886(Protozoa:Apicomplexa)by PCR in naturally infected sheep from Brazil[J].Vet Parasitol,2009,165(3-4):332-336.
[12]GJERDE B.Phylogenetic relationships among Sarcocystis species in cervids,cattle and sheep inferred from the mitochondrial cytochrome c oxidase subunit I gene[J].Int J Parasitol,2013,43(7):579-591.
[13]肖兵南,张长弓,周望平,等.几种住肉孢子虫包囊可溶性蛋白的抗原特性分析[J].畜牧兽医学报,1998,29(2):167-172.XIAO B N,ZHANG C G,ZHOU W P,et al.Analysis of antigenic characteristics of soluble proteins from several species of Sarcocystis[J].Acta Veterinaria et Zootechnica Sinica,1998,29(2):167-172.(in Chinese)
[14]SUBRAMANIAN A,MILLER D M.Structural analysis ofα-enolase.Mapping the functional domains involved in down-regulation of the c-myc protooncogene[J].J Biol Chem,2000,275(8):5958-5965.
[15]PANCHOLI V.Multifunctionalα-enolase:its role in diseases[J].Cell Mol Life Sci,2001,58(7):902-920.
[16]AVILN L,GUALDRN-LPEZ M,QUIONOSW,et al.Enolase:a key player in the metabolism and a probable virulence factor of trypanosomatid parasites-perspectives for its use as a therapeutic target[J].Enzyme Res,2011,2011:932549.
[17]DZIERSZINSKI F,MORTUAIRE M,DENDOUGAN,et al.Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii[J].J Mol Biol,2001,309(5):1017-1027.
[18]RUAN J,MOUVEAUX T,LIGHT S H,et al.The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions[J].Acta Cryst,2015,D71:417-426.
[19]WANG M,YANG X Y,ZHANG N Z,et al.Evaluation of protective immune responses induced by recombinant TrxLp and ENO2proteins against Toxoplasma gondii infection in BALB/c mice[J].Biomed Res Int,2016,2016:3571962.
[20]GUO Z G,JOHNSON A M.Genetic comparison of Neospora caninum with Toxoplasmaand Sarcocystis by random amplified polymorphic DNA-polymerase chain reaction[J].Parasitol Res,1995,81(5):365-370.
[21]YANG D Y,LIU J,HAO P,et al.MIC3,a novel cross-protective antigen expressed in Toxoplasma gondii and Neospora caninum[J].Parasitol Res,2015,114(10):3791-3799.
[22]李航,姜利建,刘晋宇,等.新孢子虫与弓形虫交叉抗原AMA1基因重组腺病毒的构建及免疫应答分析[J].中国畜牧兽医,2016,43(12):3336-3342.LI H,JIANG L J,LIU J Y,et al.Construction and analysis of immune response of Neospora caninum and Toxoplasma gondii cross recombinant adenovirus antigen AMA1gene[J].China Animal Husbandry&Veterinary Medicine,2016,43(12):3336-3342.(in Chinese)
[23]LIAO M,XUAN X,HUANG X,et al.Identification and characterization of cross-reactive antigens from Neospora caninumand Toxoplasma gondii[J].Parasitology,2005,130(5):481-488.
[24]MCALLISTER M M,PARMLEY S F,WEISS LM,et al.An immunohistochemical method for detecting bradyzoite antigen(BAG5)in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen[J].J Parasitol,1996,82(2):354-355.
[2]董辉,陆瑶瑶,杨玉荣.绵羊和山羊住肉孢子虫流行病学及分型研究进展[J].中国人兽共患病学报,2017,33(9):828-836.DONG H,LU Y Y,YANG Y R.Epidemiology and classification of Sarcocystis in sheep and goat[J].Chinese Journal of Zoonoses,2017,33(9):828-836.(in Chinese)
[3]DUBEY J P,CALERO-BERNAL R,ROSENTHALB M,et al.Sarcocystosis of animals and humans[M].2nd ed.Boca Raton:CRC Press,2016.
[4]DUBEY J P,LEEK R G,FAVER R.Prevalence,transmission,and pathogenicity of Sarcocystis gigantea of sheep[J].J Am Vet Med Assoc,1986,188(2):151-154.
[5]MUNDAY B L,OBENDORF D L.Development and growth of Sarcocystis gigantea in experimentally-infected sheep[J].Vet Parasitol,1984,15(3-4):203-211.
[6]MARTNEZ-NAVALN B,ANASTASIO-GINERB,CANO-FRUCTUOSO M,et al.Short communication.Sarcocystisinfection:a major cause of carcass condemnation in adult sheep in Spain[J].Span J Agric Res,2012,10(2):388-392.
[7]CˇERVA L,GUT J.Indirect haemagglutination reaction with antigen of Sarcocystis gigantea(Railliet,1886)Ashford,1977[J].Folia Parasitol(Praha),1983,30(3):223-228.
[8]TENTER A M.Comparison of Dot-ELISA,ELISAand IFAT for the detection of IgG antibodies to Sarcocystis muris in experimentally infected and immunized mice[J].Vet Parasitol,1988,29(2-3):89-104.
[9]MUNDAY B L.The effect of Sarcocystis ovicanis on growth rate and haematocrit in lambs[J].Vet Parasitol,1979,5(2-3):129-135.
[10]HECKEROTH A R,TENTER A M.Comparison of immunological and molecular methods for the diagnosis of infections with pathogenic Sarcocystis species in sheep[J].Tokai J Exp Clin Med,1998,23(6):293-302.
[11]DA SILVA R C,SU C L,LANGONI H.First identification of Sarcocystis tenella(Railliet,1886)Moulé,1886(Protozoa:Apicomplexa)by PCR in naturally infected sheep from Brazil[J].Vet Parasitol,2009,165(3-4):332-336.
[12]GJERDE B.Phylogenetic relationships among Sarcocystis species in cervids,cattle and sheep inferred from the mitochondrial cytochrome c oxidase subunit I gene[J].Int J Parasitol,2013,43(7):579-591.
[13]肖兵南,张长弓,周望平,等.几种住肉孢子虫包囊可溶性蛋白的抗原特性分析[J].畜牧兽医学报,1998,29(2):167-172.XIAO B N,ZHANG C G,ZHOU W P,et al.Analysis of antigenic characteristics of soluble proteins from several species of Sarcocystis[J].Acta Veterinaria et Zootechnica Sinica,1998,29(2):167-172.(in Chinese)
[14]SUBRAMANIAN A,MILLER D M.Structural analysis ofα-enolase.Mapping the functional domains involved in down-regulation of the c-myc protooncogene[J].J Biol Chem,2000,275(8):5958-5965.
[15]PANCHOLI V.Multifunctionalα-enolase:its role in diseases[J].Cell Mol Life Sci,2001,58(7):902-920.
[16]AVILN L,GUALDRN-LPEZ M,QUIONOSW,et al.Enolase:a key player in the metabolism and a probable virulence factor of trypanosomatid parasites-perspectives for its use as a therapeutic target[J].Enzyme Res,2011,2011:932549.
[17]DZIERSZINSKI F,MORTUAIRE M,DENDOUGAN,et al.Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii[J].J Mol Biol,2001,309(5):1017-1027.
[18]RUAN J,MOUVEAUX T,LIGHT S H,et al.The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions[J].Acta Cryst,2015,D71:417-426.
[19]WANG M,YANG X Y,ZHANG N Z,et al.Evaluation of protective immune responses induced by recombinant TrxLp and ENO2proteins against Toxoplasma gondii infection in BALB/c mice[J].Biomed Res Int,2016,2016:3571962.
[20]GUO Z G,JOHNSON A M.Genetic comparison of Neospora caninum with Toxoplasmaand Sarcocystis by random amplified polymorphic DNA-polymerase chain reaction[J].Parasitol Res,1995,81(5):365-370.
[21]YANG D Y,LIU J,HAO P,et al.MIC3,a novel cross-protective antigen expressed in Toxoplasma gondii and Neospora caninum[J].Parasitol Res,2015,114(10):3791-3799.
[22]李航,姜利建,刘晋宇,等.新孢子虫与弓形虫交叉抗原AMA1基因重组腺病毒的构建及免疫应答分析[J].中国畜牧兽医,2016,43(12):3336-3342.LI H,JIANG L J,LIU J Y,et al.Construction and analysis of immune response of Neospora caninum and Toxoplasma gondii cross recombinant adenovirus antigen AMA1gene[J].China Animal Husbandry&Veterinary Medicine,2016,43(12):3336-3342.(in Chinese)
[23]LIAO M,XUAN X,HUANG X,et al.Identification and characterization of cross-reactive antigens from Neospora caninumand Toxoplasma gondii[J].Parasitology,2005,130(5):481-488.
[24]MCALLISTER M M,PARMLEY S F,WEISS LM,et al.An immunohistochemical method for detecting bradyzoite antigen(BAG5)in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen[J].J Parasitol,1996,82(2):354-355.