羊巴贝斯虫未定种trap基因的克隆表达及反应原性分析
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摘要
通过克隆trap基因,在原核表达系统中进行表达并纯化,Western blot鉴定TRAP蛋白反应原性,应用生物信息学分析软件分析验证该基因/蛋白的结构。以羊巴贝斯虫未定种新疆株gDNA和cDNA为模板扩增trap基因全长;并构建pET-30a-trap原核表达载体。将构建成功的表达载体转入BL21(DE3)pLysS进行诱导表达;纯化可溶性的rBspTRAP,Western blot分析其反应原性,并应用生物信息学分析软件分析验证该基因/蛋白的结构。结果显示:获得的trap基因全长为2 338bp,开放阅读框大小为1 944bp,具有5个内含子和6个外显子。氨基酸序列分析显示1~26位氨基酸为信号肽序列,45~201位和240~288位氨基酸分别为TRAP蛋白家族的vWA和TSP1结构域。Western blot结果显示羊巴贝斯虫未定种新疆/敦煌株阳性血清可特异性识别rBspTRAP,该重组蛋白与莫氏巴贝斯虫(临潭株、天祝株、河北株、宁县株)阳性血清及尤氏泰勒虫阳性血清无交叉反应。本试验成功克隆trap基因并表达,该基因可以作为免疫学诊断用候选抗原,为以后建立鉴别诊断莫氏巴贝斯虫和羊巴贝斯虫感染的免疫学诊断方法奠定基础。
The study was conducted to clone and express trap gene of Babesia sp.(Xinjiang), analyze and verify their structures via bioinformatics softwares.The complete length of trap gene was amplified from genomic DNA and cDNA of Babesia sp.(Xinjiang), then a prokaryotic expression vector,pET-30 a-trap,was constructed and transformed into Escherichia coli BL21(DE3) pLysS to induce protein expression.Soluble rBspTRAP was purified and then its immunoreactivity was determined by Western blot. Analysis results using bioinformatics softwares showed that the trap gene is 2 338 base pairs, contained 5 introns and 6 exons with an open reading frame(ORF) of1 944 bp.The 1-23 animo acids was a signal peptide sequence and these of 45-201 and 240-288 were vWA and TSP1 motifs of TRAP protein family, respectively.The rBspTRAP could be specifically recognized by positive sera from sheep infected by Babesia sp.Xingjiang/Dunhuang.No cross-reactions were present on the sera of B.motasi Lintan/Tianzhu/Hebei/Ningxian and Theileria uilenbergi.This study successfully cloned and expressed trap gene,and rBspTRAP has the potetials for Babesia.sp and B.motasi in future.
The study was conducted to clone and express trap gene of Babesia sp.(Xinjiang), analyze and verify their structures via bioinformatics softwares.The complete length of trap gene was amplified from genomic DNA and cDNA of Babesia sp.(Xinjiang), then a prokaryotic expression vector,pET-30 a-trap,was constructed and transformed into Escherichia coli BL21(DE3) pLysS to induce protein expression.Soluble rBspTRAP was purified and then its immunoreactivity was determined by Western blot. Analysis results using bioinformatics softwares showed that the trap gene is 2 338 base pairs, contained 5 introns and 6 exons with an open reading frame(ORF) of1 944 bp.The 1-23 animo acids was a signal peptide sequence and these of 45-201 and 240-288 were vWA and TSP1 motifs of TRAP protein family, respectively.The rBspTRAP could be specifically recognized by positive sera from sheep infected by Babesia sp.Xingjiang/Dunhuang.No cross-reactions were present on the sera of B.motasi Lintan/Tianzhu/Hebei/Ningxian and Theileria uilenbergi.This study successfully cloned and expressed trap gene,and rBspTRAP has the potetials for Babesia.sp and B.motasi in future.
引文
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[20]BAUM J,PAPENFUSS A T,BAUM B,et al.Regulation of apicomplexan actin-based motility[J].Nat Rev Microbiol,2006,4(8):621-628.
[21]JONES M L,KITSON E L,RAYNER J C,et al.Plasmodium falciparum erythrocyte invasion:a conserved myosin associated complex[J].Mol Biochem Parasitol,2006,147(1):74-84.
[22]MATUSCHEWSKI K,NUNES A C,NUSSENZWEIG V,et al.Plasmodium sporozoite invasion into insect and mammalian cells is directed by the same dual binding system[J].EMBO J,2002,21(7):1597-1606.
[23]唐威华.SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因[J].植物生理学报,2000,26(1):64-68.
[2]TIAN Z C,LIU G Y,YIN H,et al.RPS8--a new informative DNA marker for phylogeny of Babesia and Theileria parasites in China[J].PLoS One,2013,8(11):e79860.
[3]GUAN G Q,LIU J L,LIU A H,et al.A member of the HSP90family from ovine Babesia in China:molecular characterization,phylogenetic analysis and antigenicity[J].Parasitology,2015,142(11):1387-1397.
[4]LIU A H,YIN H,GUAN G Q,et al.At least two genetically distinct large Babesia species infective to sheep and goats in China[J].Vet Parasitol,2007,147(3/4):246-251.
[5]苟惠天,薛慧文,殷宏,等.基于COI基因序列对我国部分巴贝斯虫分类的研究[J].畜牧兽医学报,2016,47(6):1293-1298.
[6]杨强,刘爱红,刘军龙等.我国十省份羊巴贝斯虫的分子流行病学调查[J].中国兽医科学,2016,46(5):597-601.
[7]NIU Q L,LIU Z J,YANG J F,et al.Expression of sheep pathogen Babesia sp.Xinjiang rhoptry-associated protein 1and evaluation of its diagnostic potential by enzyme-linked immunosorbent assay[J].Parasitology,2016,143(14):1990-1999.
[8]NIU Q L,LIU Z J,YANG J F,et al.Expression analysis and biological characterization of Babesia sp.BQ1(Lintan)(Babesia motasi-like)rhoptry-associated protein 1and its potential use in serodiagnosis via ELISA[J].Parasit Vectors,2016,9(1):313.
[9]KIM K,WEISS L M.Toxoplasma gondii:the model apicomplexan[J].Int J Parasitol,2004,34(3):423-432.
[10]BAUM J,PAPENFUSS A T,BAUM B,et al.Regulation of apicomplexan actin-based motility[J].Nat Rev Microbiol,2006,4(8):621-628.
[11]ZHOU J,FUKUMOTO S,JIA H,et al.Characterization of the Babesia gibsoni P18as a homologue of thrombospondin related adhesive protein[J].Mol Biochem Parasitol,2006,148(2):190-198.
[12]GOO Y K,JIA H,ABOGE G O,et al.Babesia gibsoni:Serodiagnosis of infection in dogs by an enzymelinked immunosorbent assay with recombinant BgTRAP[J].Exp Parasitol,2008,118(4):555-560.
[13]GUAN G Q,CHAUVIN A,YIN H,et al.Course of infection by Babesia sp.BQ1(Lintan)and B.divergens in sheep depends on the production of IFN-γand IL10[J].Parasite Immunol,2010,32(2):143-152.
[14]GUAN G Q,MOREAU E,LIU J,et al.BQP35is a novel member of the intrinsically unstructured protein(IUP)family which is a potential antigen for the sero-diagnosis of Babesia sp.BQ1(Lintan)infection[J].Vet Parasitol,2012,187(3/4):421-430.
[15]GOO Y K,LEE N,TERKAWI M A,et al.Development of a rapid immunochromatographic test using a recombinant thrombospondin-related adhesive protein of Babesia gibsoni[J].Vet Parasitol,2012,190(3/4):595-598.
[16]DUBREMETZ J F,GARCIA-REGUET N,CONSEIL V,et al.Apical organelles and host-cell invasion by Apicomplexa[J].Int J Parasitol,1998,28(7):1007-1013.
[17]DUBEY J P.Toxoplasma,Hammondia,Besnoitia,Sarcocystis,and other tissue cyst-forming coccidia of man and animals[J].Parasitic Protozoa,1977(3):101-237.
[18]LEVINE N D.Progress in taxonomy of the Apicomplexan protozoa[J].J Protozool,1988,35(4):518-520.
[19]MENARD R.Gliding motility and cell invasion by Apicomplexa:insights from the Plasmodium sporozoite[J].Cell Microbiol,2001,3(2):63-73.
[20]BAUM J,PAPENFUSS A T,BAUM B,et al.Regulation of apicomplexan actin-based motility[J].Nat Rev Microbiol,2006,4(8):621-628.
[21]JONES M L,KITSON E L,RAYNER J C,et al.Plasmodium falciparum erythrocyte invasion:a conserved myosin associated complex[J].Mol Biochem Parasitol,2006,147(1):74-84.
[22]MATUSCHEWSKI K,NUNES A C,NUSSENZWEIG V,et al.Plasmodium sporozoite invasion into insect and mammalian cells is directed by the same dual binding system[J].EMBO J,2002,21(7):1597-1606.
[23]唐威华.SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因[J].植物生理学报,2000,26(1):64-68.