马驽巴贝斯虫新疆伊犁株Bc48基因克隆、表达及其多克隆抗体的制备
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摘要
为表达马驽巴贝斯虫新疆伊犁株Bc48基因,制备多克隆抗体,试验根据GenBank中马驽巴贝斯虫Bc48基因序列,设计合成1对特异性引物,以提取的新疆株Bc48基因组DNA为模板进行PCR扩增,将扩增产物克隆至原核表达载体pET-28a(+)中,阳性质粒转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导后表达产物进行SDS-PAGE分析,切胶纯化蛋白后免疫6周龄雌性BALB/c小鼠,制备多克隆抗体;同时把该基因克隆至真核表达载体pCMV-N-flag中,将阳性质粒转化至大肠杆菌DH5α感受态细胞中,提取pCMV-N-flag-Bc48质粒转染到293T细胞中进行真核表达。结果显示,获得了大小约为15ku的融合蛋白,与预期目的蛋白大小相一致;免疫BALB/c小鼠制备的多克隆抗体经ELISA和Western blotting检测、验证,能特异地识别相应抗原,其抗体效价可达1∶128 000,说明该多克隆抗体有较高的抗体效价和特异性;真核表达质粒在293T细胞中表达Bc48蛋白。本试验可为进一步研究马驽巴贝斯虫功能基因及建立快速的检测方法奠定基础,在虫株的分类学研究及候选疫苗的研制中有重要意义。
In order to express Bc48 gene of Babesia caballi from Xinjiang Yili,and prepare its polyclonal antibody,apair of specific primers was designed according to Bc48 gene sequence of Babesia caballi in GenBank.The genomic DNA was extracted from Babesia caballi,and Bc48 gene was cloned into pET-28 a(+)expression vector.The positive plasmid was transformed to Escherichia coli BL21(DE3).After IPTG induction,the product was analyzed by SDS-PAGE,six weeks old female BALB/c mice were immunized with purified Bc48 protein to prepare polyclonal antibodies.Meanwhile Bc48 gene was cloned into eukaryotic expression vector pCMV-N-flag.The positive plasmid was transformed to Escherichia coli DH5α,eukaryotic expression of pCMV-Nflag-Bc48 plasmid was transfected into 293 Tcells.The results showed that the recombinant pro-tein with 15 ku was obtained,which was consistent with the expected protein size;Western blotting analysis showed that polyclonal antibodies could specifically identify the corresponded antigens;ELISA analysis showed that the antibodies had high titer(1∶128 000);And eukaryotic expression plasmids expressed Bc48 protein in 293 Tcells.This test could lay the foundation for further study on the functional genes and establish quick detection method of Babesia caballi.It had important significance in the study of strain taxonomy and the development of candidate vaccine.
In order to express Bc48 gene of Babesia caballi from Xinjiang Yili,and prepare its polyclonal antibody,apair of specific primers was designed according to Bc48 gene sequence of Babesia caballi in GenBank.The genomic DNA was extracted from Babesia caballi,and Bc48 gene was cloned into pET-28 a(+)expression vector.The positive plasmid was transformed to Escherichia coli BL21(DE3).After IPTG induction,the product was analyzed by SDS-PAGE,six weeks old female BALB/c mice were immunized with purified Bc48 protein to prepare polyclonal antibodies.Meanwhile Bc48 gene was cloned into eukaryotic expression vector pCMV-N-flag.The positive plasmid was transformed to Escherichia coli DH5α,eukaryotic expression of pCMV-Nflag-Bc48 plasmid was transfected into 293 Tcells.The results showed that the recombinant pro-tein with 15 ku was obtained,which was consistent with the expected protein size;Western blotting analysis showed that polyclonal antibodies could specifically identify the corresponded antigens;ELISA analysis showed that the antibodies had high titer(1∶128 000);And eukaryotic expression plasmids expressed Bc48 protein in 293 Tcells.This test could lay the foundation for further study on the functional genes and establish quick detection method of Babesia caballi.It had important significance in the study of strain taxonomy and the development of candidate vaccine.
引文
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[5]FRIEDHOFF K T,TENTER A M.Haemoparasites of equines:Impact on international trade of horses[J].Revue Scientifique Et Technique,1990,9(4):1187-1194.
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[7]罗金,刘光远,谢俊仁,等.驽巴贝斯虫病PCR检测方法的建立和应用[J].中国畜牧兽医,2011,38(7):193-195.LUO J,LIU G Y,XIE J R,et al.Establishment and application of Babesia caballi PCR detection method[J].China Animal Husbandry&Veterinary Medicine,2011,38(7):193-195.(in Chinese)
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[10]赵祥平,王玉玲,侯艳梅.驽巴贝斯虫病巢式PCR检测方法的研究[J].中国动物检疫,2009,26(10):37-38.ZHAO X P,WANG Y L,HOU Y M.A study on the detection method of Babesia caballi in the nested PCR[J].Chinese Animal Quarantine,2009,26(10):37-38.(in Chinese)
[11]王群,郑小龙,朱来华,等.马媾疫锥虫SYBR GreenⅠ荧光定量PCR检测方法的建立[J].中国预防兽医学报,2012,34(9):724-727.WANG Q,ZHENG X L,ZHU L H,et al.Establishment of Trypanosoma equiperdum SYBR GreenⅠReal-time fluorescence quantitative PCR method[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(9):724-727.(in Chinese)
[12]FRIEDHOFF K T.Piroplasmas of horses——Impact on the international horse trade[J].Berl Munch Tierarztl Wochenschr,1982,95(19):368-374.
[13]IKADAI H,XUAN X,IGARASHI I,et al.Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay[J].Journal of Clinical Microbiology,1999,37(11):3475-3480.
[14]BSE R,PEYMANN B.Diagnosis of Babesia caballi infections in horses by enzyme-linked immunosorbent assay(ELISA)and Western blot[J].International Journal for Parasitology,1994,24(3):347-356.
[15]SAM YELLOWE T Y.Rhoptry organelles of the apicomplexa:Their role in host cell invasion and intracellular survival[J].Parasitol Today,1996,12(8):308-316.
[16]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.GAO S Y,CHA E H,WANG K,et al.A method of purifying prokaryotic expression protein with high cost performance SDS-PAGE cutting[J].Chinese Agricultural Science Bulletin,2010,26(22):24-26.(in Chinese)
[17]吕绘倩,蒋洁兰,姜志强,等.太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备[J].大连海洋大学学报,2017,32(2):127-133.LV H Q,JIANG J L,JIANG Z Q,et al.Prokaryotic expression of the antifreeze gene and preparation of polyclonal antibody[J].Journal of Dalian Fisheries University,2017,32(2):127-133.(in Chinese)
[18]MEHLHORN H,SCHEIN E.Redescription of Babesia equi Laveran,1901as Theileria equi Mehlhorn,Schein 1998[J].Parasitology Research,1998,84(6):467-475.
[19]UILENBERG G.Babesia——A historical overview[J].Veterinary Parasitology,2006,138(1-2):3-10.
[20]张杨.马驽巴贝斯虫二温式、荧光PCR检测方法的建立及其初步应用[D].乌鲁木齐:新疆农业大学,2015.ZHANG Y.Establishment of a two temperature PCR assay and Real-time fluorescence quantitative PCR method for detection of Babesia caballi[D].Urumqi:Xinjiang Agricultural University,2015.(in Chinese)
[21]SCHWINT O N,UETI M W,PALMER G H,et al.Imidocarb dipropionate clears persistent Babesia caballi infection with elimination of transmission potential[J].Antimicrobial Agents&Chemotherapy,2009,53(10):4327-4332.
[22]薛书江,于龙政,曹世诺,等.驽巴贝虫BC-48基因的克隆及其在大肠杆菌中的表达[J].中国兽医科学,2007,37(3):214-217.XUE S J,YU L Z,CAO S N,et al.Cloning and expression of BC-48gene of Babesia caballi Jilin strain and its expression in Escherichia coli[J].Chinese Veterinary Science,2007,37(3):214-217.(in Chinese)
[2]MACHADO R Z,TOLEDO C Z,TEIXEIRA M C,et al.Molecular and serological detection of Theileria equi and Babesia caballi in donkeys(Equus asinus)in Brazil[J].Veterinary Parasitology,2012,186(3-4):461-465.
[3]BRUNING A,PHIPPS P,POSNETT E,et al.Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis[J].Veterinary Parasitology,1997,68(1-2):11-26.
[4]IKADAI H,TAMAKI Y,XUAN X,et al.Inhibitory effect of monoclonal antibodies on the growth of Babesia caballi[J].International Journal for Parasitology,1999,29(11):1785-1791.
[5]FRIEDHOFF K T,TENTER A M.Haemoparasites of equines:Impact on international trade of horses[J].Revue Scientifique Et Technique,1990,9(4):1187-1194.
[6]BASHIRUDDIN J B,CAMMA C,REBELO E.Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the16SrRNA gene[J].Veterinary Parasitology,1999,84(1-2):75-83.
[7]罗金,刘光远,谢俊仁,等.驽巴贝斯虫病PCR检测方法的建立和应用[J].中国畜牧兽医,2011,38(7):193-195.LUO J,LIU G Y,XIE J R,et al.Establishment and application of Babesia caballi PCR detection method[J].China Animal Husbandry&Veterinary Medicine,2011,38(7):193-195.(in Chinese)
[8]LI Y,TENG L,SHANG L,et al.Detection of Theileriaand Babesiasp in Ixodid ticks from Qinghai province,Northwestern China[J].Journal of Animal&Veterinary Advances,2013,12(6):775-778.
[9]薛书江,于龙政,曹世诺,等.驽巴贝斯虫PCR检测方法的建立及应用[J].河南农业科学,2007,4:106-109.XUE S J,YU L Z,CAO S N,et al.Establishment and application of Babesia cballi PCR detection method[J].Journal of Henan Agricultural Sciences,2007,4:106-109.(in Chinese)
[10]赵祥平,王玉玲,侯艳梅.驽巴贝斯虫病巢式PCR检测方法的研究[J].中国动物检疫,2009,26(10):37-38.ZHAO X P,WANG Y L,HOU Y M.A study on the detection method of Babesia caballi in the nested PCR[J].Chinese Animal Quarantine,2009,26(10):37-38.(in Chinese)
[11]王群,郑小龙,朱来华,等.马媾疫锥虫SYBR GreenⅠ荧光定量PCR检测方法的建立[J].中国预防兽医学报,2012,34(9):724-727.WANG Q,ZHENG X L,ZHU L H,et al.Establishment of Trypanosoma equiperdum SYBR GreenⅠReal-time fluorescence quantitative PCR method[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(9):724-727.(in Chinese)
[12]FRIEDHOFF K T.Piroplasmas of horses——Impact on the international horse trade[J].Berl Munch Tierarztl Wochenschr,1982,95(19):368-374.
[13]IKADAI H,XUAN X,IGARASHI I,et al.Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay[J].Journal of Clinical Microbiology,1999,37(11):3475-3480.
[14]BSE R,PEYMANN B.Diagnosis of Babesia caballi infections in horses by enzyme-linked immunosorbent assay(ELISA)and Western blot[J].International Journal for Parasitology,1994,24(3):347-356.
[15]SAM YELLOWE T Y.Rhoptry organelles of the apicomplexa:Their role in host cell invasion and intracellular survival[J].Parasitol Today,1996,12(8):308-316.
[16]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.GAO S Y,CHA E H,WANG K,et al.A method of purifying prokaryotic expression protein with high cost performance SDS-PAGE cutting[J].Chinese Agricultural Science Bulletin,2010,26(22):24-26.(in Chinese)
[17]吕绘倩,蒋洁兰,姜志强,等.太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备[J].大连海洋大学学报,2017,32(2):127-133.LV H Q,JIANG J L,JIANG Z Q,et al.Prokaryotic expression of the antifreeze gene and preparation of polyclonal antibody[J].Journal of Dalian Fisheries University,2017,32(2):127-133.(in Chinese)
[18]MEHLHORN H,SCHEIN E.Redescription of Babesia equi Laveran,1901as Theileria equi Mehlhorn,Schein 1998[J].Parasitology Research,1998,84(6):467-475.
[19]UILENBERG G.Babesia——A historical overview[J].Veterinary Parasitology,2006,138(1-2):3-10.
[20]张杨.马驽巴贝斯虫二温式、荧光PCR检测方法的建立及其初步应用[D].乌鲁木齐:新疆农业大学,2015.ZHANG Y.Establishment of a two temperature PCR assay and Real-time fluorescence quantitative PCR method for detection of Babesia caballi[D].Urumqi:Xinjiang Agricultural University,2015.(in Chinese)
[21]SCHWINT O N,UETI M W,PALMER G H,et al.Imidocarb dipropionate clears persistent Babesia caballi infection with elimination of transmission potential[J].Antimicrobial Agents&Chemotherapy,2009,53(10):4327-4332.
[22]薛书江,于龙政,曹世诺,等.驽巴贝虫BC-48基因的克隆及其在大肠杆菌中的表达[J].中国兽医科学,2007,37(3):214-217.XUE S J,YU L Z,CAO S N,et al.Cloning and expression of BC-48gene of Babesia caballi Jilin strain and its expression in Escherichia coli[J].Chinese Veterinary Science,2007,37(3):214-217.(in Chinese)