单链抗体对Cry1A类毒素的检测方法建立及识别差异初步研究
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摘要
为建立一种针对Cry1类毒素的经济高效的检测方法,本研究以杂交瘤细胞株2D10 cDNA为模板,通过重叠延伸PCR(gene splicing by overlap extension PCR, SOE-PCR)技术构建单链抗体(single-chain variable fragment, scFv)基因,转入大肠杆菌(Escherichia coli) BL21 (DE3)表达并建立双抗体夹心酶联免疫吸附测定(double antibody sandwich ELISA, DAS-ELISA)检测方法,通过分子对接模拟分析影响scFv与Cry1A类毒素结合的关键因素。结果成功构建了scFv基因,表达纯化出具有较高识别活性的scFv抗体(约28 kD),所建立的DAS-ELISA方法对Cry1Ab/Cry1Ac毒素的最低检测限(limits of determination, LOD)为20 ng/mL,分子对接结果显示,毒素的三维结构决定了其结合活性,氢键和疏水作用力在scFv与毒素结合过程中起重要作用。本研究利用基因工程抗体技术构建并表达获得了scFv抗体,建立了针对Cry1Ab/Cry1Ac毒素的双抗夹心ELISA检测方法,初步分析研究了scFv与Cry1Ac/Cry1Ab和Cry1Aa的识别差异机制,为进一步改造、研究开发新型广谱scFv提供了理论依据。
To establish a cost-effective detection method for Cry1 toxins, a hybridoma cell line 2D10 was used as a c DNA template to construct a single-chain antibody(scFv) gene via gene splicing by overlap extension PCR(SOE-PCR). After being transferred into Escherichia coli BL21(DE3), the scFv gene was expressed and a double antibody sandwich ELISA(DAS-ELISA) was established. Meanwhile, the key factors affecting the interaction of scFv to Cry1 A toxins were analyzed by molecular docking simulation. As a result,the scFv gene was successfully constructed and the scFv antibody with high activity was purified(about 28kD). The minimum detection limit(LOD) of the established DAS-ELISA for Cry1Ab/Cry1 Ac toxin was 20 ng/mL. The docking results showed that the three-dimensional structure of the toxin determined its binding activity, hydrogen bonds and hydrophobic interaction played an important role during the binding process between scFv and Cry1 A toxins. In this study, a scFv was constructed and expressed based on genetic engineering antibody technology, a DAS-ELISA was established for Cry1 Ab/Cry1 Ac toxin determination and the recognization mechanism difference of scFv to Cry1 Ac/Cry1 Ab and Cry1 Aa was preliminarily analyzed which provide a theoretical basis for the research and development of a new broad-spectrum scFv.
To establish a cost-effective detection method for Cry1 toxins, a hybridoma cell line 2D10 was used as a c DNA template to construct a single-chain antibody(scFv) gene via gene splicing by overlap extension PCR(SOE-PCR). After being transferred into Escherichia coli BL21(DE3), the scFv gene was expressed and a double antibody sandwich ELISA(DAS-ELISA) was established. Meanwhile, the key factors affecting the interaction of scFv to Cry1 A toxins were analyzed by molecular docking simulation. As a result,the scFv gene was successfully constructed and the scFv antibody with high activity was purified(about 28kD). The minimum detection limit(LOD) of the established DAS-ELISA for Cry1Ab/Cry1 Ac toxin was 20 ng/mL. The docking results showed that the three-dimensional structure of the toxin determined its binding activity, hydrogen bonds and hydrophobic interaction played an important role during the binding process between scFv and Cry1 A toxins. In this study, a scFv was constructed and expressed based on genetic engineering antibody technology, a DAS-ELISA was established for Cry1 Ab/Cry1 Ac toxin determination and the recognization mechanism difference of scFv to Cry1 Ac/Cry1 Ab and Cry1 Aa was preliminarily analyzed which provide a theoretical basis for the research and development of a new broad-spectrum scFv.
引文
孙硕,张坤,谭桂玉,等.2013.双抗夹心酶免疫法检测转Bt基因抗虫棉种子的研究[J].棉花学报,25(1):45-50.(Sun S,Zhang K,Tan G Y,et al.2013.Determination of Bt protein in transgenic cotton seeds by double antibody sandwich enzyme-linked immunosorbent assay[J].Cotton Science,25(1):45-50.)
王克夷.1999.疏水作用和蛋白质[J].生命的化学,19(5):233-235.(Wang K Y.1999.Hydrophobic Interaction and Protein[J].Chemistry of Life,19(5):233-235.)
武爱华,王耘,刘媛,等.2017.磁珠筛选抗Cry2Aa人源化单链抗体及检测方法的建立[J].江苏农业学报,33(4):945-950.(Wu A H,Wang Y,Liu Y,et al.2017.Screening and detection of Cry2Aa-binding specific single chain antibody fragments(scFv)from a humanized phage display library by magnetic beads[J].Jiangsu Journal of Agriculture Science,33(4):945-950.)
谢小波,舒庆.2001.用Envirologix Cry1Ab/Cry1Ac试剂盒快速测定转基因水稻Bt杀虫蛋白含量的研究[J].中国农业科学,34(5):465-468.(Xie X B,Shu Q.2001.Studies on rapid quantitative analysis of Bt toxin by using envirologix kits in transgenic rice[J].Scientia Agricultura Sinica,34(5):465-468.)
徐重新,张存政,张霄,等.2013.人源化抗Cry1B毒素蛋白单链抗体的原核表达及生物学活性测定[J].南京农业大学学报,36(3):47-52.(Xu C X,Zhang C Z,Zhang X,et al.2013.Eecretory expression and characterization of humanized anti-Cry1B scFv antibody in Escherichia coli[J].Journal of Nanjing Agricultural University,36(3):47-52).
郑蓉,吕暾.2011.毒素DON单链抗体的同源建模及与DON结合的分子模拟研究[J].化学学报,69(23):2882-2888.(Zheng R,Lv D.2011.Homology modeling of anti-DON Scfv antibody and molecular simulation study between scFv and its antigen DON[J].Acta Chimica Sinica,69(23):2882-2888.)
Bravo A,Likitvivatanavong S,Gill S S,et al.2011.Bacillus thuringiensis:A story of a successful bio-insecticide[J].Insect Biochemistry and Molecular Biology,41:423-431.
Bravo A,Soberón M.2008.How to cope with insect resistance to Bt toxins?[J].Trends in Biotechnology,26(10):573-579.
Dong S,Zhang C,Zhang X,et al.2016.Production and characterization of monoclonal antibody broadly recognizing Cry1 toxins by use of designed polypeptide as hapten[J].Anayitical Chemistry,88:7023-7032.
Karsunke X Z,Wang H,Weber E,et al.2012.Developmen of single-chain variable fragment(scFv)antibodies against hapten benzo[a]pyrene:A binding study[J].Analytical and Bioanalytical Chemistry,402:499-507.
Kaur S.2012.Risk assessment of Bt transgenic crops[J].Bacillus thuringiensis Biotechnology,41-85.
Kumar R.2012.A real-time immuno-PCR assay for the detection of transgenic Cry1Ab protein[J].European Food Research and Technology,234:101-108.
Li J,Huang R,Xia K,et al.2014.Double antibodies sandwich enzyme-linked immunosorbent assay for the detection of Alicyclobacillus acidoterrestris in apple juice concentrate[J].Food Control,40(2014):172-176.
Lorenzen N,Olesen N J,J?rgensen P E V.1990.Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein[J]Journal of General Virology,71(3):561-567.
Saxena D,Flores S,Stotzky G.1999.Transgenic plants insecticidal toxin in root exudates from Bt corn[J].Nature402(6761):480-481.
Wark K L,Hudson P J.2006.Latest technologies for the enhancement of antibody affinity[J].Advanced Drug Delivery Reviews,58:657-670.
Wyatt G M,Garrett S D,Lee H A,et al.1999.Alteration of the binding characteristics of a recombinant scFv antiparathion antibody-1.Mutagenesis targeted at the V(H)CDR3 domain[J].Food and Agricultural Immunology,11:207-218.
Xin L,Li P,Qi Z,et al.2012.Molecular Characterization of monoclonal antibodies against aflatoxins:A possible explanation for the highest sensitivity[J].Analytical Chemistry,84(12):5229.
Yu H L,Li Y H,Wu K M.2011.Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non-target organisms[J].Journal of Integrative Plant Biology,53(7).
Zhang X,Xu C,Zhang C,et al.2014.Established a new double antibodies sandwich enzymelinked immunosorbent assay for detecting Bacillus thuringiensis(Bt)Cry1Ab toxin based single-chain variable fragments from a na?ve mouse phage displayed library[J].Toxin,81:13-22.
王克夷.1999.疏水作用和蛋白质[J].生命的化学,19(5):233-235.(Wang K Y.1999.Hydrophobic Interaction and Protein[J].Chemistry of Life,19(5):233-235.)
武爱华,王耘,刘媛,等.2017.磁珠筛选抗Cry2Aa人源化单链抗体及检测方法的建立[J].江苏农业学报,33(4):945-950.(Wu A H,Wang Y,Liu Y,et al.2017.Screening and detection of Cry2Aa-binding specific single chain antibody fragments(scFv)from a humanized phage display library by magnetic beads[J].Jiangsu Journal of Agriculture Science,33(4):945-950.)
谢小波,舒庆.2001.用Envirologix Cry1Ab/Cry1Ac试剂盒快速测定转基因水稻Bt杀虫蛋白含量的研究[J].中国农业科学,34(5):465-468.(Xie X B,Shu Q.2001.Studies on rapid quantitative analysis of Bt toxin by using envirologix kits in transgenic rice[J].Scientia Agricultura Sinica,34(5):465-468.)
徐重新,张存政,张霄,等.2013.人源化抗Cry1B毒素蛋白单链抗体的原核表达及生物学活性测定[J].南京农业大学学报,36(3):47-52.(Xu C X,Zhang C Z,Zhang X,et al.2013.Eecretory expression and characterization of humanized anti-Cry1B scFv antibody in Escherichia coli[J].Journal of Nanjing Agricultural University,36(3):47-52).
郑蓉,吕暾.2011.毒素DON单链抗体的同源建模及与DON结合的分子模拟研究[J].化学学报,69(23):2882-2888.(Zheng R,Lv D.2011.Homology modeling of anti-DON Scfv antibody and molecular simulation study between scFv and its antigen DON[J].Acta Chimica Sinica,69(23):2882-2888.)
Bravo A,Likitvivatanavong S,Gill S S,et al.2011.Bacillus thuringiensis:A story of a successful bio-insecticide[J].Insect Biochemistry and Molecular Biology,41:423-431.
Bravo A,Soberón M.2008.How to cope with insect resistance to Bt toxins?[J].Trends in Biotechnology,26(10):573-579.
Dong S,Zhang C,Zhang X,et al.2016.Production and characterization of monoclonal antibody broadly recognizing Cry1 toxins by use of designed polypeptide as hapten[J].Anayitical Chemistry,88:7023-7032.
Karsunke X Z,Wang H,Weber E,et al.2012.Developmen of single-chain variable fragment(scFv)antibodies against hapten benzo[a]pyrene:A binding study[J].Analytical and Bioanalytical Chemistry,402:499-507.
Kaur S.2012.Risk assessment of Bt transgenic crops[J].Bacillus thuringiensis Biotechnology,41-85.
Kumar R.2012.A real-time immuno-PCR assay for the detection of transgenic Cry1Ab protein[J].European Food Research and Technology,234:101-108.
Li J,Huang R,Xia K,et al.2014.Double antibodies sandwich enzyme-linked immunosorbent assay for the detection of Alicyclobacillus acidoterrestris in apple juice concentrate[J].Food Control,40(2014):172-176.
Lorenzen N,Olesen N J,J?rgensen P E V.1990.Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein[J]Journal of General Virology,71(3):561-567.
Saxena D,Flores S,Stotzky G.1999.Transgenic plants insecticidal toxin in root exudates from Bt corn[J].Nature402(6761):480-481.
Wark K L,Hudson P J.2006.Latest technologies for the enhancement of antibody affinity[J].Advanced Drug Delivery Reviews,58:657-670.
Wyatt G M,Garrett S D,Lee H A,et al.1999.Alteration of the binding characteristics of a recombinant scFv antiparathion antibody-1.Mutagenesis targeted at the V(H)CDR3 domain[J].Food and Agricultural Immunology,11:207-218.
Xin L,Li P,Qi Z,et al.2012.Molecular Characterization of monoclonal antibodies against aflatoxins:A possible explanation for the highest sensitivity[J].Analytical Chemistry,84(12):5229.
Yu H L,Li Y H,Wu K M.2011.Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non-target organisms[J].Journal of Integrative Plant Biology,53(7).
Zhang X,Xu C,Zhang C,et al.2014.Established a new double antibodies sandwich enzymelinked immunosorbent assay for detecting Bacillus thuringiensis(Bt)Cry1Ab toxin based single-chain variable fragments from a na?ve mouse phage displayed library[J].Toxin,81:13-22.