大肠杆菌Fts Z蛋白原核表达及多克隆抗体的制备与鉴定
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摘要
为制备特异性抗大肠杆菌丝状热敏蛋白Z(Escherichia coli filamentous thermosensitive protein Z,Ec-FtsZ)多克隆抗体,将Ec-FtsZ基因进行化学合成后连接pET-22b(+)表达载体,构建重组质粒Ec-FtsZ-pET-22b(+)。将重组质粒转化到大肠杆菌E.coli BL21(DE3)中进行Ec-FtsZ原核表达与表达条件优化,以HisTrap层析柱进行Ec-FtsZ的分离纯化,再以孔雀绿法进行Ec-FtsZ GTPase(Guanosine triphosphatase)活性测定。使用纯化的Ec-FtsZ为抗原免疫大鼠制备多克隆抗体,经酶联免疫吸附测定实验(Enzyme-linked immunosorbent assay,ELISA)、Western blotting实验和免疫荧光实验鉴定,抗Ec-FtsZ多克隆抗体效价可达1∶256 000且具有良好的抗原特异性。抗Ec-FtsZ多克隆抗体的成功制备为Ec-FtsZ生物学功能研究和生化检测奠定了实验基础。
To prepare polyclonal antibody(PcAb) against Escherichia coli filamentous thermosensitive protein Z(Ec-FtsZ),the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22 b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase(Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay(ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared,which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.
To prepare polyclonal antibody(PcAb) against Escherichia coli filamentous thermosensitive protein Z(Ec-FtsZ),the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22 b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase(Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay(ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared,which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.
引文
[1]Zhang W,Yang ZJ,Chen DJ.The research progress of FtsZ inhibitor,a target for bacteria cell-division.Chin Bull Life Sci,2012,22(1):94-101(in Chinese).张雯,杨志钧,陈代杰.靶向FtsZ的细胞分裂抑制剂的研究进展.生命科学,2012,22(1):94-101.
[2]Krupka M,Margolin W.Unite to divide:oligomerization of tubulin and actin homologs regulates initiation of bacterial cell division.F1000Res,2018,7:235.
[3]Sang Y,Tao J,Yao YF.Regulation of the Z ring positioning in bacterial cell division-A review.Acta Microbiol Sin,2013,53(4):321-327(in Chinese).桑昱,陶晶,姚玉峰.细菌分裂Z环定位的调控方式.微生物学报,2013,53(4):321-327.
[4]Lin Y,Zhu NY,Jiang JD,et al.The establishment and application of a high-throughput screening(HTS)assay for inhibitors of Mycobacterium tuberculosis FtsZ.Chin J Antibiot,2015,40(3):166-170,212(in Chinese).林媛,朱宁屿,蒋建东,等.以FtsZ为靶点的抗结核药物筛选模型的建立及应用.中国抗生素杂志,2015,40(3):166-170,212.
[5]Erickson HP,Anderson DE,Osawa M.FtsZ in bacterial cytokinesis:cytoskeleton and force generator all in one.Microbiol Mol Biol Rev,2010,74(4):504-528.
[6]Ortiz C,Natale P,Cueto L,et al.The keepers of the ring:regulators of FtsZ assembly.FEMS Microbiol Rev,2016,40(1):57-67.
[7]Yang SY,Zhu J,Wu Y,et al.Purification and crystallization of MinC/FtsZ complex in Escherichia coli.Chemistry,2017,80(1):89-93(in Chinese).杨少媛,朱嘉,吴彦,等.大肠杆菌MinC/FtsZ蛋白复合物的纯化和结晶.化学通报,2017,80(1):89-93.
[8]Kapoor S,Panda D.Targeting FtsZ for antibacterial therapy:a promising avenue.Expert Opin Ther Targets,2009,13(9):1037-1051.
[9]Lin Y,Zhu NY,Han YX,et al.Identification of anti-tuberculosis agents that target the cell-division protein FtsZ.J Antibiot(Tokyo),2014,67(9):671-676.
[10]Lin Y,Zhang HJ,Zhu NY,et al.Identification of TB-E12 as a novel FtsZ inhibitor with anti-tuberculosis activity.Tuberculosis,2018,110:79-85.
[11]Zhang QS,Yuan RL,Jing SR.Expression and purification of Norovirus capsid protein VP1 and preparation of the polyclonal antibodies.Chin J Microecol,2017,29(8):896-899,905(in Chinese).仉秋实,苑荣亮,井申荣.诺如病毒NV衣壳蛋白VP1表达纯化及多克隆抗体的制备.中国微生态学杂志,2017,29(8):896-899,905.
[12]Chen YY,Niu XY,Lin Y,et al.Prokaryotic expression,purification and preparation of rat polyclonal antibody against Escherichia coli ZipA.Chin J Cell Mol Immunol,2018,34(10):942-948(in Chinese).陈云雨,牛夏忆,林媛,等.大肠杆菌丝状热敏蛋白Z相互作用蛋白A(Ec-ZipA)原核表达、纯化与大鼠多克隆抗体的制备.细胞与分子免疫学杂志,2018,34(10):942-948.
[13]Ray S,Jindal B,Kunal K,et al.BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly.FEBS J,2015,282(20):4015-4033.
[14]Artola M,Ruiz-Avila LB,Vergo?ós A,et al.Effective GTP-replacing FtsZ inhibitors and antibacterial mechanism of action.ACS Chem Biol,2015,10(3):834-843.
[15]Xiao J,Goley ED.Redefining the roles of the FtsZ-ring in bacterial cytokinesis.Curr Opin Microbiol,2016,34:90-96.
[16]Panda D,Bhattacharya D,Gao QH,et al.Identification of agents targeting FtsZ assembly.Future Med Chem,2016,8(10):1111-1132.
[17]Wu FF,Cao ZG,Wang Q,et al.Prokaryotic expression of Zaire Ebola virus GP and the preparation of polyclonal antibody.Chin J Vet Sci,2018,38(3):496-501(in Chinese).吴芳芳,曹增国,王琪,等.扎伊尔型埃博拉病毒糖蛋白的原核表达及多克隆抗体的制备.中国兽医学报,2018,38(3):496-501.
[18]Sun T,Yang GW,Zhang JY,et al.Prokaryotic expression of Hepatitis C Virus(HCV)NS3 protein and preparation of polyclonal antibody.Chin J Biotech,2015,31(5):711-721(in Chinese).孙涛,杨光文,张金阳,等.丙型肝炎病毒NS3蛋白的原核表达及多克隆抗体制备.生物工程学报,2015,31(5):711-721.
[19]Wang H,Yang B,Zhao KS,et al.Preparation and identification of polyclonal antibodies against Moraxella catarrhalis UspA1.Chin J Biotech,2018,34(1):102-109(in Chinese).王辉,杨波,赵可胜,等.卡他莫拉菌UspA1蛋白多克隆抗体的制备及鉴定.生物工程学报,2018,34(1):102-109.
[20]Qin YJ,Zhang TH,Ye X.Preparation and detection of anti-influenza A virus polymerase basic protein 1polyclonal antibody.Chin J Biotech,2016,32(1):105-113(in Chinese).秦玉洁,张廷虹,叶昕.抗A型流感病毒聚合酶碱性蛋白1多克隆抗体的制备和鉴定.生物工程学报,2016,32(1):105-113.
[21]Hayat SMG,Farahani N,Golichenari B,et al.Recombinant protein expression in Escherichia coli(E.coli):What we need to know.Curr Pharm Des,2018,24(6):718-725.
[22]Baeshen MN,Al-Hejin AM,Bora RS,et al.Production of biopharmaceuticals in E.coli:Current scenario and future perspectives.J Microbiol Biotechnol,2015,25(7):953-962.
[23]Papaneophytou CP,Kontopidis G.Statistical approaches to maximize recombinant protein expression in Escherichia coli:A general review.Protein Expr Purif,2014,94:22-32.
[24]Su P,Gong GL.Research progress on optimizing the expression of exogenous proteins in Escherichia coli.Biotechnol Bull,2017,33(2):16-23(in Chinese).苏鹏,龚国利.优化大肠杆菌表达外源蛋白的研究进展.生物技术通报,2017,33(2):16-23.
[25]Overton TW.Recombinant protein production in bacterial hosts.Drug Discov Today,2014,19(5):590-601.
[26]Haranahalli K,Tong S,Ojima I.Recent advances in the discovery and development of antibacterial agents targeting the cell-division protein FtsZ.Bioorg Med Chem,2016,24(24):6354-6369.
[2]Krupka M,Margolin W.Unite to divide:oligomerization of tubulin and actin homologs regulates initiation of bacterial cell division.F1000Res,2018,7:235.
[3]Sang Y,Tao J,Yao YF.Regulation of the Z ring positioning in bacterial cell division-A review.Acta Microbiol Sin,2013,53(4):321-327(in Chinese).桑昱,陶晶,姚玉峰.细菌分裂Z环定位的调控方式.微生物学报,2013,53(4):321-327.
[4]Lin Y,Zhu NY,Jiang JD,et al.The establishment and application of a high-throughput screening(HTS)assay for inhibitors of Mycobacterium tuberculosis FtsZ.Chin J Antibiot,2015,40(3):166-170,212(in Chinese).林媛,朱宁屿,蒋建东,等.以FtsZ为靶点的抗结核药物筛选模型的建立及应用.中国抗生素杂志,2015,40(3):166-170,212.
[5]Erickson HP,Anderson DE,Osawa M.FtsZ in bacterial cytokinesis:cytoskeleton and force generator all in one.Microbiol Mol Biol Rev,2010,74(4):504-528.
[6]Ortiz C,Natale P,Cueto L,et al.The keepers of the ring:regulators of FtsZ assembly.FEMS Microbiol Rev,2016,40(1):57-67.
[7]Yang SY,Zhu J,Wu Y,et al.Purification and crystallization of MinC/FtsZ complex in Escherichia coli.Chemistry,2017,80(1):89-93(in Chinese).杨少媛,朱嘉,吴彦,等.大肠杆菌MinC/FtsZ蛋白复合物的纯化和结晶.化学通报,2017,80(1):89-93.
[8]Kapoor S,Panda D.Targeting FtsZ for antibacterial therapy:a promising avenue.Expert Opin Ther Targets,2009,13(9):1037-1051.
[9]Lin Y,Zhu NY,Han YX,et al.Identification of anti-tuberculosis agents that target the cell-division protein FtsZ.J Antibiot(Tokyo),2014,67(9):671-676.
[10]Lin Y,Zhang HJ,Zhu NY,et al.Identification of TB-E12 as a novel FtsZ inhibitor with anti-tuberculosis activity.Tuberculosis,2018,110:79-85.
[11]Zhang QS,Yuan RL,Jing SR.Expression and purification of Norovirus capsid protein VP1 and preparation of the polyclonal antibodies.Chin J Microecol,2017,29(8):896-899,905(in Chinese).仉秋实,苑荣亮,井申荣.诺如病毒NV衣壳蛋白VP1表达纯化及多克隆抗体的制备.中国微生态学杂志,2017,29(8):896-899,905.
[12]Chen YY,Niu XY,Lin Y,et al.Prokaryotic expression,purification and preparation of rat polyclonal antibody against Escherichia coli ZipA.Chin J Cell Mol Immunol,2018,34(10):942-948(in Chinese).陈云雨,牛夏忆,林媛,等.大肠杆菌丝状热敏蛋白Z相互作用蛋白A(Ec-ZipA)原核表达、纯化与大鼠多克隆抗体的制备.细胞与分子免疫学杂志,2018,34(10):942-948.
[13]Ray S,Jindal B,Kunal K,et al.BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly.FEBS J,2015,282(20):4015-4033.
[14]Artola M,Ruiz-Avila LB,Vergo?ós A,et al.Effective GTP-replacing FtsZ inhibitors and antibacterial mechanism of action.ACS Chem Biol,2015,10(3):834-843.
[15]Xiao J,Goley ED.Redefining the roles of the FtsZ-ring in bacterial cytokinesis.Curr Opin Microbiol,2016,34:90-96.
[16]Panda D,Bhattacharya D,Gao QH,et al.Identification of agents targeting FtsZ assembly.Future Med Chem,2016,8(10):1111-1132.
[17]Wu FF,Cao ZG,Wang Q,et al.Prokaryotic expression of Zaire Ebola virus GP and the preparation of polyclonal antibody.Chin J Vet Sci,2018,38(3):496-501(in Chinese).吴芳芳,曹增国,王琪,等.扎伊尔型埃博拉病毒糖蛋白的原核表达及多克隆抗体的制备.中国兽医学报,2018,38(3):496-501.
[18]Sun T,Yang GW,Zhang JY,et al.Prokaryotic expression of Hepatitis C Virus(HCV)NS3 protein and preparation of polyclonal antibody.Chin J Biotech,2015,31(5):711-721(in Chinese).孙涛,杨光文,张金阳,等.丙型肝炎病毒NS3蛋白的原核表达及多克隆抗体制备.生物工程学报,2015,31(5):711-721.
[19]Wang H,Yang B,Zhao KS,et al.Preparation and identification of polyclonal antibodies against Moraxella catarrhalis UspA1.Chin J Biotech,2018,34(1):102-109(in Chinese).王辉,杨波,赵可胜,等.卡他莫拉菌UspA1蛋白多克隆抗体的制备及鉴定.生物工程学报,2018,34(1):102-109.
[20]Qin YJ,Zhang TH,Ye X.Preparation and detection of anti-influenza A virus polymerase basic protein 1polyclonal antibody.Chin J Biotech,2016,32(1):105-113(in Chinese).秦玉洁,张廷虹,叶昕.抗A型流感病毒聚合酶碱性蛋白1多克隆抗体的制备和鉴定.生物工程学报,2016,32(1):105-113.
[21]Hayat SMG,Farahani N,Golichenari B,et al.Recombinant protein expression in Escherichia coli(E.coli):What we need to know.Curr Pharm Des,2018,24(6):718-725.
[22]Baeshen MN,Al-Hejin AM,Bora RS,et al.Production of biopharmaceuticals in E.coli:Current scenario and future perspectives.J Microbiol Biotechnol,2015,25(7):953-962.
[23]Papaneophytou CP,Kontopidis G.Statistical approaches to maximize recombinant protein expression in Escherichia coli:A general review.Protein Expr Purif,2014,94:22-32.
[24]Su P,Gong GL.Research progress on optimizing the expression of exogenous proteins in Escherichia coli.Biotechnol Bull,2017,33(2):16-23(in Chinese).苏鹏,龚国利.优化大肠杆菌表达外源蛋白的研究进展.生物技术通报,2017,33(2):16-23.
[25]Overton TW.Recombinant protein production in bacterial hosts.Drug Discov Today,2014,19(5):590-601.
[26]Haranahalli K,Tong S,Ojima I.Recent advances in the discovery and development of antibacterial agents targeting the cell-division protein FtsZ.Bioorg Med Chem,2016,24(24):6354-6369.
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