抗对硫磷单链抗体在昆虫细胞中的表达及活性鉴定
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摘要
利用Bac-to-Bac杆状病毒表达系统,在Sf9昆虫细胞中表达抗对硫磷单链抗体,并评价该重组抗体sc Fv-4C6的分子识别活性。以分泌能特异性识别对硫磷的单克隆抗体的杂交瘤细胞株4C6为RNA来源,采用RT-PCR方法扩增抗体的重链和轻链可变区基因,经重叠延伸PCR方法串联拼接获得单链抗体基因片段(sc Fv-4C6)。构建包含目的片段的重组杆粒Bacmidsc Fv-4C6,转染Sf9细胞表达目的蛋白,采用免疫印迹法(Western blotting)检测表达产物,间接竞争酶联免疫吸附(ic-ELISA)法评价产物的生物活性。结果表明:sc Fv-4C6基因片段拼装正确,成功转染Sf9细胞,并在转染后72 h表达量最高,表达的单链抗体大小为28.3 k D;表达产物能特异性识别对硫磷,IC50值为7.9 ng/m L,对甲基对硫磷和杀螟硫磷分别有12%和1.8%的交叉反应率,与亲本单克隆抗体的识别性能相似。该研究表明,具有生物活性的抗对硫磷单链抗体sc Fv-4C6可在昆虫细胞中成功得到表达。
In this study, sc Fv-coding genes were obtained from a hybridoma cell and expressed in Bacto-Bac baculovirus expression system, and the characterizations of sc Fv were verified by Western blotting, ELISA and compared with monoclonal antibody. Firstly, the specific heavy and light variable chains were amplified from hybridoma cell line 4C6, which secreting monoclonal antibody(m Ab)against parathion-ethyl, and then they were assembled as sc Fv fragments by splice overlap extension polymerase chain reaction(SOE-PCR). Thereafter, the bacmid-sc Fv-4C6 was constructed and transfected into Sf9 insect cells to generate the recombinant antibody which was further verified by Western blotting. The results demonstrated that the recombinant sc Fv-4C6, with the relative molecular mass of approximately 28.3 k D, was successfully constructed and expressed in Sf9 cells. Besides, the expression peak was reached at 72 h after the transfection. As tested by ic-ELISA, the performances of sc Fv-4C6 were similar to those of the parental m Ab, with a high sensitivity(IC50 value) of 7.9 ng/m L to parathion-ethyl, 12% cross reactivity to parathion-methyl, and 1.8% cross reactivity to fenitrothion.
In this study, sc Fv-coding genes were obtained from a hybridoma cell and expressed in Bacto-Bac baculovirus expression system, and the characterizations of sc Fv were verified by Western blotting, ELISA and compared with monoclonal antibody. Firstly, the specific heavy and light variable chains were amplified from hybridoma cell line 4C6, which secreting monoclonal antibody(m Ab)against parathion-ethyl, and then they were assembled as sc Fv fragments by splice overlap extension polymerase chain reaction(SOE-PCR). Thereafter, the bacmid-sc Fv-4C6 was constructed and transfected into Sf9 insect cells to generate the recombinant antibody which was further verified by Western blotting. The results demonstrated that the recombinant sc Fv-4C6, with the relative molecular mass of approximately 28.3 k D, was successfully constructed and expressed in Sf9 cells. Besides, the expression peak was reached at 72 h after the transfection. As tested by ic-ELISA, the performances of sc Fv-4C6 were similar to those of the parental m Ab, with a high sensitivity(IC50 value) of 7.9 ng/m L to parathion-ethyl, 12% cross reactivity to parathion-methyl, and 1.8% cross reactivity to fenitrothion.
引文
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[23]LEMEULLE C,CHARDèS T,MONTAVON C,et al.Antidigoxin sc Fv fragments expressed in bacteria and in insect cells have different antigen binding properties[J].FEBS Lett,1998,423(2):159-166.
[24]徐重新,张霄,张存政,等.抗Bt(Cry1B)毒素单链抗体在sf9昆虫细胞中的表达及活性测定[J].江苏农业学报,2013,29(5):985-991.XU C X,ZHANG X,ZHANG C Z,et al.Expression and characterization of anti-Bt(Cry1B)toxin sc Fv antibody in sf9insect cells[J].Jiangsu J Agric Sci,2013,29(5):985-991.
[25]XU Z L,DONG J X,WANG H,et al.Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay[J].J Agric Food Chem,2012,60(20):5076-5083.
[26]HORá?EK J,GARRETT S D,SKLáDAL P,et al.Characterization of the interactions between immobilized parathion and the corresponding recombinant sc Fv antibody using a piezoelectric biosensor[J].Food Agric Immunol,1998,10(4):363-374.
[27]乔亚奇,潘家荣,王磊,等.抗对硫磷基因工程新型抗体的制备及鉴定[J].微生物学报,2010,50(5):670-675.QIAO Y Q,PAN J R,WANG L,et al.Construction and identification of genetically engineered tetravalent antibodies against parathion[J].Acta Microbiologica Sinica,2010,50(5):670-675.
[28]张付凯,乔亚奇,王磊,等.抗对硫磷基因工程四价抗体在大肠杆菌中高效表达与鉴定[J].核农学报,2012,26(1):118-122.ZHANG F K,QIAO Y Q,WANG L,et al.High level expression of genetically engineered tetravalent antibodies against parathion in E.coli and its identification[J].J Nucl Agric Sci,2012,26(1):118-122.(in Chinese)
[29]YAP W N.Development of novel galactosylation method for the expression of recombinant human transferrin in insect cell culture[D].Malaysia:Universiti Teknologi Malaysia,2006.
[30]JANG Y J,STOLLAR B D.Anti-DNA antibodies:aspects of structure and pathogenicity[J].Cell Mol Life Sci,2003,60(2):309-320.
[31]LIN Y,LI B,WANG M,et al.Improved affinity of a chicken single-chain antibody to avian infectious bronchitis virus by sitedirected mutagenesis of complementarity-determining region H3[J].African J Biotechnol,2011,10(79):18294-18303.
[2]MOSTAFALOU S,ABDOLLAHI M.Pesticides and human chronic diseases:Evidences,mechanisms,and perspectives[J].Toxicol Appl Pharm,2013,268(2):157-177.
[3]VELASCO A,HERNáNDEZ S,RAMíREZ M,et al.Detection of residual organochlorine and organophosphorus pesticides in agricultural soil in Rio Verde region of San Luis Potosi,Mexico[J].J Environ Sci Heal B,2014,49(7):498-504.
[4]YU R,LIU Q,LIU J S,et al.Concentrations of organophosphorus pesticides in fresh vegetables and related human health risk assessment in Changchun,Northeast China[J].Food Control,2016,60:353-360.
[5]VELKOSKA-MARKOVSKA L,PETANOVSKA-ILIEVSKA B.Optimization and development of a SPE-HPLC-DAD method for the determination of atrazine,malathion,fenitrothion,and parathion pesticide residues in apple juice[J].Maced J Chem Chem En,2013,32(2):299-308.
[6]YAN X,SHI H Y,WANG M H.Development of an enzymelinked immunosorbent assay for the simultaneous determination of parathion and imidacloprid[J].Anal Methods-UK,2012,4(12):4053-4057.
[7]邓浩,孔德彬,杨金易,等.对硫磷化学发光酶联免疫吸附分析方法的建立和评价[J].分析化学,2013,41(2):247-252.DENG H,KONG D B,YANG J Y,et al.Development of an indirect competitive chemiluminescence enzyme-linked immunoassay for parathion[J].Chin J Anal Chem,2013,41(2):247-252.
[8]LIU Y H,XIE R,GUO Y R,et al.Comparison of homologous and heterologous formats in nanocolloidal gold-based immunoassays for parathion residue determination[J].J Environ Sci Health B,2012,47(5):475-483.
[9]FUNARI R,DELLA VENTURA B,SCHIAVO L,et al.Detection of parathion pesticide by quartz crystal microbalance functionalized with UV-activated antibodies[J].Anal Chem,2013,85(13):6392-6397.
[10]FUNARI R,DELLA VENTURA B,CARRIERI R,et al.Detection of parathion and patulin by quartz-crystal microbalance functionalized by the photonics immobilization technique[J].Biosens Bioelectron,2015,67:224-229.
[11]XUE C G,NAN T G,WU S Q,et al.Detection of ethyl parathion using microcantilever biosensor[J].Asian J Chem,2012,24(9):3951-3953.
[12]MARKUS V,JANNE L,URPO L.Directed antibody-engineering techniques and their applications in food immunoassays[J].Trac Trends Analc Chem,2011,30(2):219-226.
[13]LI X,LI P W,ZHANG Q,et al.Molecular characterization of monoclonal antibodies against aflatoxins:a possible explanation for the highest sensitivity[J].Anal Chem,2012,84(12):5229-5235.
[14]WYATT G M,GARRETT S D,LEE H A,et al.Alteration of the binding characteristics of a recombinant sc Fv anti-parathion antibody-1.Mutagenesis targeted at the VH CDR3 domain[J].Food Agric Immunol,1999,11(3):207-218.
[15]KARSUNKE X Y Z,WANG H F,WEBER E,et al.Development of single-chain variable fragment(sc Fv)antibodies against hapten benzo[α]pyrene:a binding study[J].Anal Bioanal Chem,2012,402(1):499-507.
[16]SCHIRRMANN T,AL-HALABI L,DüBEL S,et al.Production systems for recombinant antibodies[J].Front Biosci,2008,13:4576-4594.
[17]STECH M,MERK H,SCHENK J A,et al.Production of functional antibody fragments in a vesicle-based eukaryotic cellfree translation system[J].J Biotech,2013,164(2):220-231.
[18]VERMA R,BOLETI E,GEORGE A J T.Antibody engineering:comparison of bacterial,yeast,insect and mammalian expression systems[J].J Immunol Methods,1998,216(1-2):165-181.
[19]LI X,LI P W,LEI J W,et al.A simple strategy to obtain ultrasensitive single-chain fragment variable antibodies for aflatoxin detection[J].RSC Adv,2013,3(44):22367-22372.
[20]ZHANG X,LIU Y,ZHANG C Z,et al.Rapid isolation of singlechain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis(Bt)Cry1B toxin[J].Ecotox Environ Safe,2012,81:84-90.
[21]GARRETT S D,APPLEFORD D J A,WYATT G M,et al.Production of a recombinant anti-parathion antibody(sc Fv);Stability in methanolic food extracts and comparison to an antiparathion monoclonal antibody[J].J Agric Food Chem,1997,45(10):4183-4189.
[22]MORENO M J,PLANA E,MANCLúS J J,et al.Comparative study of monoclonal and recombinant antibody-based immunoassays for fungicide analysis in fruit juices[J].Food Anal Method,2014,7(2):481-489.
[23]LEMEULLE C,CHARDèS T,MONTAVON C,et al.Antidigoxin sc Fv fragments expressed in bacteria and in insect cells have different antigen binding properties[J].FEBS Lett,1998,423(2):159-166.
[24]徐重新,张霄,张存政,等.抗Bt(Cry1B)毒素单链抗体在sf9昆虫细胞中的表达及活性测定[J].江苏农业学报,2013,29(5):985-991.XU C X,ZHANG X,ZHANG C Z,et al.Expression and characterization of anti-Bt(Cry1B)toxin sc Fv antibody in sf9insect cells[J].Jiangsu J Agric Sci,2013,29(5):985-991.
[25]XU Z L,DONG J X,WANG H,et al.Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay[J].J Agric Food Chem,2012,60(20):5076-5083.
[26]HORá?EK J,GARRETT S D,SKLáDAL P,et al.Characterization of the interactions between immobilized parathion and the corresponding recombinant sc Fv antibody using a piezoelectric biosensor[J].Food Agric Immunol,1998,10(4):363-374.
[27]乔亚奇,潘家荣,王磊,等.抗对硫磷基因工程新型抗体的制备及鉴定[J].微生物学报,2010,50(5):670-675.QIAO Y Q,PAN J R,WANG L,et al.Construction and identification of genetically engineered tetravalent antibodies against parathion[J].Acta Microbiologica Sinica,2010,50(5):670-675.
[28]张付凯,乔亚奇,王磊,等.抗对硫磷基因工程四价抗体在大肠杆菌中高效表达与鉴定[J].核农学报,2012,26(1):118-122.ZHANG F K,QIAO Y Q,WANG L,et al.High level expression of genetically engineered tetravalent antibodies against parathion in E.coli and its identification[J].J Nucl Agric Sci,2012,26(1):118-122.(in Chinese)
[29]YAP W N.Development of novel galactosylation method for the expression of recombinant human transferrin in insect cell culture[D].Malaysia:Universiti Teknologi Malaysia,2006.
[30]JANG Y J,STOLLAR B D.Anti-DNA antibodies:aspects of structure and pathogenicity[J].Cell Mol Life Sci,2003,60(2):309-320.
[31]LIN Y,LI B,WANG M,et al.Improved affinity of a chicken single-chain antibody to avian infectious bronchitis virus by sitedirected mutagenesis of complementarity-determining region H3[J].African J Biotechnol,2011,10(79):18294-18303.