泰妙菌素人工完全抗原的制备及其单克隆抗体的研制
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摘要
目的研制灵敏度高、特异性好的抗泰妙菌素(TML)单克隆抗体。方法用肟化法和碳二亚胺法将TML分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联,制备免疫原TML-BSA和包被原TML-OVA,用TML-BSA免疫BALB/c小鼠,经细胞融合并采用间接ELISA筛选抗TML杂交瘤细胞株,用体内诱生法制备腹水和辛酸-硫酸铵法纯化获得单克隆抗体,并用SDS-PAGE鉴定。结果成功筛选出2株杂交瘤细胞株3B3和4A7,其细胞上清效价分别为1∶1600和1∶6400。纯化后单克隆抗体4A7的效价为5.12×10~5,半数抑制剂量(IC_(50))为0.049 ng/mL,亲和力常数Kaff为1.47×10~9 L/mol,与类似物瑞他帕林有2.7%的交叉反应率,与沃尼妙林、氟罗沙星等其他抗生素均无交叉反应。结论成功研制了抗TML单克隆抗体。
Objective To develop high-sensitivity and high-specificity monoclonal antibody against tiamulin(TML). Methods Using oximation and carbodiimide method, TML was conjugated to bovine serum albumin(BSA) and ovalbumin(OVA) to prepare the immunogen TML-BSA and coating antigen TML-OVA, respectively. BALB/c mice were immunized with TML-BSA. The anti-TML monoclonal antibody hybridoma cell lines were screened by indirect ELISA after cell fusion. The ascites were prepared by in vivo induction method. The monoclonal antibody was purified from ascites by caprylic acid-ammonium sulfate method and identified by SDS-PAGE. Results The hybridoma cell lines of 3B3 and 4A7 were successfully obtained. Their titers of cell supernatant were 1∶1600 and 1∶6400, respectively. After purification, the titer of monoclonal antibody 4A7 was 5.12×10~5. The half inhibitory concentration(IC_(50)) of 4A7 was 0.049 ng/mL. The affinity constant Kaff of 4A7 was 1.47×10~9 L/mol. The cross-reaction rate of 4A7 was 2.7% with analog retapamulin, whereas no cross-reaction was detected with other antibiotics such as valnemulin and fleroxacin. Conclusion Anti-TML monoclonal antibody has been successfully developed.
Objective To develop high-sensitivity and high-specificity monoclonal antibody against tiamulin(TML). Methods Using oximation and carbodiimide method, TML was conjugated to bovine serum albumin(BSA) and ovalbumin(OVA) to prepare the immunogen TML-BSA and coating antigen TML-OVA, respectively. BALB/c mice were immunized with TML-BSA. The anti-TML monoclonal antibody hybridoma cell lines were screened by indirect ELISA after cell fusion. The ascites were prepared by in vivo induction method. The monoclonal antibody was purified from ascites by caprylic acid-ammonium sulfate method and identified by SDS-PAGE. Results The hybridoma cell lines of 3B3 and 4A7 were successfully obtained. Their titers of cell supernatant were 1∶1600 and 1∶6400, respectively. After purification, the titer of monoclonal antibody 4A7 was 5.12×10~5. The half inhibitory concentration(IC_(50)) of 4A7 was 0.049 ng/mL. The affinity constant Kaff of 4A7 was 1.47×10~9 L/mol. The cross-reaction rate of 4A7 was 2.7% with analog retapamulin, whereas no cross-reaction was detected with other antibiotics such as valnemulin and fleroxacin. Conclusion Anti-TML monoclonal antibody has been successfully developed.
引文
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[4] Walczak K,Friendship R,Brockoff E,et al.Treatment rates for injectable tiamulin and lincomycin as an estimate of morbidity in a swineherd with endemic swine dysentery[J].Can Vet J,2017,58(5):472-481.
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[9] 王凤芹,杨媛媛,陈楚昊,等.固相萃取-超高效液相色谱-串联质谱法测定饲料中沃尼妙林和泰妙菌素[J].动物营养学报,2018,30(6):2394-2401.Wang F,Yang Y,Chen C,et al.Determination of valnemulin and tiamulin in feed by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry[J].Dong Wu Ying Yang Xue Bao,2018,30(6):2394-2401.
[10] 郭林云,苏亚,于海东,等.超高效液相色谱-串联质谱法测定鸡蛋中泰妙菌素残留[J].内蒙古石油化工,2016,42(6):34-36.Guo L,Su Y,Yu Ha,et al.Determination of tiamulin residues in eggs by ultraperformance liquid chromatography-tandem mass spectrometry[J].Neimenggu Shi You Hua Gong,2016,42(6):34-36.
[11] Sun F,Yang S,Zhang H,et al.Comprehensive analysis of tiamulin metabolites in various species of farm animals using ultra-high-performance liquid chromatography coupled to quadrupole/time-of-flight 9[J].J Agric Food Chem,2017,65(1):199-207.
[12] Jansen L J M,Bolck Y J C,Rademaker J,et al.The analysis of tetracyclines,quinolones,macrolides,lincosamides,pleuromutilins,and sulfonamides in chicken feathers using UHPLC-MS/MS in order to monitor antibiotic use in the poultry sector[J].Anal Bioanal Chem,2017,409(21):4927-4941.
[13] Xiao X,Sun J,Yang T,et al.Pharmacokinetic/pharmacodynamic profiles of tiamulin in an experimental intratracheal infection model of Mycoplasma gallisepticum[J/OL].Front Vet Sci,2016,3:75.DOI:10.3389/fvets.2016.00075.eCollection 2016.
[14] Patyra E,Nebot C,Gavilan R E,et al.Development and validation of an LC-MS/MS method for the quantification of tiamulin,trimethoprim,tylosin,sulfadiazine and sulfamethazine in medicated feed[J].Food Addit Contam Part A Chem Anal Control Expo Risk Assess,2018,3(5):882-891.
[15] Saita T,Yamamoto Y,Hosoya K,et al.An ultra-specific and sensitive sandwich ELISA for imatinib using two anti-imatinib antibodies[J].Anal Chim Acta,2017,969:72-78.
[16] Krittanai S,Kitisripanya T,Udomsin O,et al.Development of a colloidal gold nanoparticle-based immunochromatographic strip for the one-step detection of miroestrol and puerarin[J/OL].Biomed Chromatogr,2018,32(11):e4330.DOI:10.1002/bmc.4330.Epub 2018 Aug 15.
[17] Dong G,Pan Y,Wang Y,et al.Preparation of a broad-spectrum anti-zearalenone and its primary analogues antibody and its application in an indirect competitive enzyme-linkedimmunosorbent assay[J].Food Chem,2018,247:8-15.
[18] 成磊,吴海珍,费静,等.4-戊基苯酚单克隆抗体的制备及其可变区的同源建模[J].细胞与分子免疫学杂志,2017,33(3):390-396.Cheng L,Wu H,Fei J,et al.Preparation of monoclonal antibody with 4-pentylphenol and homology modeling of its variable region[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2017,33(3):390-396.
[19] 戴华,陈俊华,张海霞,等.抗鸡白细胞介素4单克隆抗体的制备及其鉴定[J].细胞与分子免疫学杂志,2017,33(7):986-990.Dai H,Chen J,Zhang H,et al.Preparation and identification of anti-chicken interleukin-4 monoclonal antibody[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2017,33(7):986-990.
[20] 赵向绒,张海祥,刘杨,等.His标签单克隆抗体的制备及交叉抗原的表位分析[J].细胞与分子免疫学杂志,2016,32(5):683-687.Zhao X,Zhang H,Liu Y,et al.Preparation of His-tag monoclonal antibody and epitope analysis of cross-antigens[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2016,32(5):683-687.
[21] Peng D,Wei W,Pan Y,et al.Preparation of a monoclonal antibody against amantadine and rimantadine and development of an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken muscle and liver[J].J Pharm Biomed Anal,2017,133:56-63.
[22] Yi S,Fu Y,Dong P,et al.Synthesis and biological activity evaluation of novel heterocyclic pleuromutilin derivative[J/OL].Molecules,2017,22(6):E996.DOI:10.3390/molecules22060996.
[23] Yi Y,Fu Y,Wang K,et al.Synthesis and antibacterial activities of novel pleuromutilin derivatives[J/OL].Arch Pharm (Weinheim),2018,351(9):e1800155.DOI:10.1002/ardp.201800155.Epub 2018 Jul 29.
[24] 衣云鹏.截短侧耳素类衍生物的合成、 结构鉴定与生物活性研究[D].中国农业科学院,2018:12-13.Yi Y.Synthesis,structure identification and biological activity of pleuromutilin derivatives[D].Zhongguo Nong Ye Ke Xue Yuan,2018:12-13.
[25] 匡华,雷咸禄,胥传来,等.一株泰妙菌素单克隆抗体杂交瘤细胞株及其应用[P].江苏:CN108330102A,2018-07-27.Yan H,Lei X,Qi C,et al.A tiamulin monoclonal antibody hybridoma cell line and its application[P].Jiangsu:CN108330102A,2018-07-27.
[26] 王新洲.泰妙菌素完全抗原的制备及其多克隆抗体的研制[D].郑州大学,2014:24-26.Wang X.Preparation of tiamulin complete antigen and preparation of polyclonal antibody[D].Zhengzhou University,2014:24-26.
[27] Lei X,Song S,Tao H,et al.Development of indirect competitive enzyme-linked immunosorbent and immunochromatographic strip assays for tiamulin detection in chicken[J/OL].ACS Omega,2018,3:3581-3586.(2018-3-28)[2019-01-16].https://www.x-mol.com/paper/616783.
[2] 刘东良,贾海燕,魏建超,等.截短侧耳类药物耐药机制研究进展[J].畜牧与兽医,2017,49(2):107-111.Liu D,Jia H,Wei J,et al.Advances in research on drug resistance mechanism of truncated collaterals[J].Xu Mu Yu Shou Yi,2017,49(2):107-111.
[3] Delic N,Draskovic V,Stevanovic J,et al.The efficacy of two phytogenic feed additives in the control of swine dysentery[J].Acta Vet,2018,68(2):178-189.
[4] Walczak K,Friendship R,Brockoff E,et al.Treatment rates for injectable tiamulin and lincomycin as an estimate of morbidity in a swineherd with endemic swine dysentery[J].Can Vet J,2017,58(5):472-481.
[5] Singh S B,Young K,Silver L L.What is an “ideal” antibiotic?Discovery challenges and path forward[J].Biochem Pharmacol,2017,133:63-73.
[6] 范宏.LC-MS/MS法测定猪肉中泰妙菌素及8-α-羟基泰妙菌素[J].食品研究与开发,2017,38(16):139-143.Fan H.Determination of tiamulin and 8-α-hydroxy tiamulin in pork by LC-MS/MS method[J].Shi Pin Yan Jiu Yu Kai Fa,2017,38(16):139-143.
[7] 国家治理监督检验检疫总局进出口食品安全局.动物性食品中兽药最高残留限量(农业部2002年235号公告)[S/OL].(2002-12-24)[2019-01-21].http://jckspaqj.aqsiq.gov.cn/dwyxspjyjy/gnxgbz/200610/t20061027_9809.htm.General Administration of Quality Supervision,Inspection and Quarantine of the People’s Repubic of China.Maximum residue limits for veterinary drugs in animal food (Announcement No.235 of the Ministry of Agriculture,2002)[S/OL].(2002-12-24)[2019-01-21].http://jckspaqj.aqsiq.gov.cn/dwyxspjyjy/gnxgbz/200610/t20061027_9809.htm.
[8] 郭紫峰,胡晶晶.液相色谱法测定饲料中泰妙菌素含量[J].黑龙江畜牧兽医,2017(7):284-285.Guo Z,Hu J.Determination of tiamulin in feed by liquid chromatography[J].Heilongjiang Xu Mu Shou Yi,2017(7):284-285.
[9] 王凤芹,杨媛媛,陈楚昊,等.固相萃取-超高效液相色谱-串联质谱法测定饲料中沃尼妙林和泰妙菌素[J].动物营养学报,2018,30(6):2394-2401.Wang F,Yang Y,Chen C,et al.Determination of valnemulin and tiamulin in feed by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry[J].Dong Wu Ying Yang Xue Bao,2018,30(6):2394-2401.
[10] 郭林云,苏亚,于海东,等.超高效液相色谱-串联质谱法测定鸡蛋中泰妙菌素残留[J].内蒙古石油化工,2016,42(6):34-36.Guo L,Su Y,Yu Ha,et al.Determination of tiamulin residues in eggs by ultraperformance liquid chromatography-tandem mass spectrometry[J].Neimenggu Shi You Hua Gong,2016,42(6):34-36.
[11] Sun F,Yang S,Zhang H,et al.Comprehensive analysis of tiamulin metabolites in various species of farm animals using ultra-high-performance liquid chromatography coupled to quadrupole/time-of-flight 9[J].J Agric Food Chem,2017,65(1):199-207.
[12] Jansen L J M,Bolck Y J C,Rademaker J,et al.The analysis of tetracyclines,quinolones,macrolides,lincosamides,pleuromutilins,and sulfonamides in chicken feathers using UHPLC-MS/MS in order to monitor antibiotic use in the poultry sector[J].Anal Bioanal Chem,2017,409(21):4927-4941.
[13] Xiao X,Sun J,Yang T,et al.Pharmacokinetic/pharmacodynamic profiles of tiamulin in an experimental intratracheal infection model of Mycoplasma gallisepticum[J/OL].Front Vet Sci,2016,3:75.DOI:10.3389/fvets.2016.00075.eCollection 2016.
[14] Patyra E,Nebot C,Gavilan R E,et al.Development and validation of an LC-MS/MS method for the quantification of tiamulin,trimethoprim,tylosin,sulfadiazine and sulfamethazine in medicated feed[J].Food Addit Contam Part A Chem Anal Control Expo Risk Assess,2018,3(5):882-891.
[15] Saita T,Yamamoto Y,Hosoya K,et al.An ultra-specific and sensitive sandwich ELISA for imatinib using two anti-imatinib antibodies[J].Anal Chim Acta,2017,969:72-78.
[16] Krittanai S,Kitisripanya T,Udomsin O,et al.Development of a colloidal gold nanoparticle-based immunochromatographic strip for the one-step detection of miroestrol and puerarin[J/OL].Biomed Chromatogr,2018,32(11):e4330.DOI:10.1002/bmc.4330.Epub 2018 Aug 15.
[17] Dong G,Pan Y,Wang Y,et al.Preparation of a broad-spectrum anti-zearalenone and its primary analogues antibody and its application in an indirect competitive enzyme-linkedimmunosorbent assay[J].Food Chem,2018,247:8-15.
[18] 成磊,吴海珍,费静,等.4-戊基苯酚单克隆抗体的制备及其可变区的同源建模[J].细胞与分子免疫学杂志,2017,33(3):390-396.Cheng L,Wu H,Fei J,et al.Preparation of monoclonal antibody with 4-pentylphenol and homology modeling of its variable region[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2017,33(3):390-396.
[19] 戴华,陈俊华,张海霞,等.抗鸡白细胞介素4单克隆抗体的制备及其鉴定[J].细胞与分子免疫学杂志,2017,33(7):986-990.Dai H,Chen J,Zhang H,et al.Preparation and identification of anti-chicken interleukin-4 monoclonal antibody[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2017,33(7):986-990.
[20] 赵向绒,张海祥,刘杨,等.His标签单克隆抗体的制备及交叉抗原的表位分析[J].细胞与分子免疫学杂志,2016,32(5):683-687.Zhao X,Zhang H,Liu Y,et al.Preparation of His-tag monoclonal antibody and epitope analysis of cross-antigens[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2016,32(5):683-687.
[21] Peng D,Wei W,Pan Y,et al.Preparation of a monoclonal antibody against amantadine and rimantadine and development of an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken muscle and liver[J].J Pharm Biomed Anal,2017,133:56-63.
[22] Yi S,Fu Y,Dong P,et al.Synthesis and biological activity evaluation of novel heterocyclic pleuromutilin derivative[J/OL].Molecules,2017,22(6):E996.DOI:10.3390/molecules22060996.
[23] Yi Y,Fu Y,Wang K,et al.Synthesis and antibacterial activities of novel pleuromutilin derivatives[J/OL].Arch Pharm (Weinheim),2018,351(9):e1800155.DOI:10.1002/ardp.201800155.Epub 2018 Jul 29.
[24] 衣云鹏.截短侧耳素类衍生物的合成、 结构鉴定与生物活性研究[D].中国农业科学院,2018:12-13.Yi Y.Synthesis,structure identification and biological activity of pleuromutilin derivatives[D].Zhongguo Nong Ye Ke Xue Yuan,2018:12-13.
[25] 匡华,雷咸禄,胥传来,等.一株泰妙菌素单克隆抗体杂交瘤细胞株及其应用[P].江苏:CN108330102A,2018-07-27.Yan H,Lei X,Qi C,et al.A tiamulin monoclonal antibody hybridoma cell line and its application[P].Jiangsu:CN108330102A,2018-07-27.
[26] 王新洲.泰妙菌素完全抗原的制备及其多克隆抗体的研制[D].郑州大学,2014:24-26.Wang X.Preparation of tiamulin complete antigen and preparation of polyclonal antibody[D].Zhengzhou University,2014:24-26.
[27] Lei X,Song S,Tao H,et al.Development of indirect competitive enzyme-linked immunosorbent and immunochromatographic strip assays for tiamulin detection in chicken[J/OL].ACS Omega,2018,3:3581-3586.(2018-3-28)[2019-01-16].https://www.x-mol.com/paper/616783.