多效唑和热变性Bt Cry1Ac免疫检测技术及其应用研究
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摘要
多效唑是广泛应用于控制植物株高的植物生长调节剂,具有常温条件下在土壤中两年不分解的高残留特性,本研究旨在建立灵敏高效的残留检测监控方法并研究其实际应用。转基因安全受到广泛关注,苏云金芽孢杆菌杀虫晶体蛋白Bt Cry1Ac是我国诸多抗虫转基因蛋白中商业化应用最广的,因为缺乏抗体,传统的免疫检测方法对于在生产加工中热变性的蛋白无效。本研究旨在探索热变性Bt Cry1Ac蛋白的单克隆抗体制备技术,为今后建立实用免疫检测方法提供方法基础。
本研究制备了多效唑半抗原,并偶联蛋白制备得到完全抗原。制备了多效唑的单克隆抗体,并建立了多效唑间接竞争酶联免疫检测方法。研究发明了热变性Bt Cry1Ac蛋白免疫原的制备方法,通过免疫新西兰大耳兔及BALB/c小鼠分别制备了其多克隆抗血清和单克隆抗体,并建立热变性Bt CrylAc蛋白的双抗夹心免疫检测方法。
取得的研究结果和结论如下：
(1)制备得到多效唑的单克隆抗体McAb6H73C9株系,并建立了间接竞争酶联免疫检测方法(icELISA)。该icELISA的50%抑制浓度(IC50)值为8.7ng/g,标准曲线的工作范围为2.0-50.4ng/mL。在小麦籽粒中的添加回收率为84.3-118.9%,变异系数为3.9-14.2%。使用建立的icELISA检测实际样品发现最大的残留浓度为0.07mg/Kg,实验证明多效唑残留能够从喷施多效唑的幼苗转移到籽粒中,结果通过了液相色谱-电喷雾四级杆轨道阱质谱(LC-ESI-MS)验证,两种方法的相关系数0.979。这说明建立的icELISA的灵敏度足以检测小麦籽粒中的多效唑残留。
(2)首次成功制备了变性Bt CrylAc的免疫原。使用热变性的方式,将溶解在0.01M,pH=7.5的PBS中的1.0mg/mL Bt Cry1Ac蛋白在100℃C沸水浴处理5min,与弗氏完全佐剂或者不完全佐剂同体积混合乳化即为免疫原。分别使用包被蛋白ELISA和兔多抗-蛋白-鼠抗夹心ELISA的方式筛选得到了对非变性和热变性Bt Cry1Ac有阳性的杂交瘤。得到特异性识别包被的非变Bt Cry1Ac的杂交瘤株系为1F2和3C9,单克隆5E9C6、5E9D8、3E6A3、3E6A5和3E6E2分泌的抗体能特异性地识别热变性Bt CrylAc。5E9C6和3E6E2能够分泌专用于Western Blot的抗体。为今后快速制备免疫印迹用鼠单抗提供了技术经验。
(3)小分子化合物和大分子蛋白的单克隆抗体制备技术原理相同,但单克隆株系的筛选方法不同。小分子单克隆株系筛选主要通过间接竞争酶联免疫检测法检测细胞培养上清液中的阳性和抑制效果。研究发现,蛋白质吸附在固相载体上时空间构象与其在溶液中是不同的,这可能改变了抗原决定簇的构象。所以活性蛋白质的单克隆株系筛选应该通过双抗夹心的方法,变性蛋白和应用于免疫印迹抗体的筛选,可以通过热变性免疫原制备。
Paclobutrazol is a widely used plant growth regulator which could reduce the height of plants. The residue of paclobutrazol won't degradation for more than two years in the applied soil at room temperature. This research was to develop a sensitive and effective method for residue detection and monitor, and study the practical application. The safety was a much concern on transgenic proteins. Bacillus thuringiensis Bt CrylAc is one of the most commercial used insecticidal proteins in China. The common ELISA could not be used to detect heat denatured protein due to lack of antibody. This research has developed monoclonal antibody of heat denatured Bt Cry1Ac, which is a method basis for the development of practical enzyme linked immunosorbent assay (ELISA).
The hapten of paclobutrazol was synthesized. Complete antigen was obtained by conjugating hapten to BSA/OVA The indirect competitive enzyme linked immunosorbent assay (icELISA) for paclobutrazol was developed based on the obtained monoclonal antibody (McAb). The immunogen of heat denatured Bt CrylAc was invented. Rabbit antiserum and mouse monoclonal antibody were obtained by the immunization of New Zealand rabbit and BALB/c mouse, respectively. And the sandwich ELISA for heat denatured Bt CrylAc was developed
The main results as follows:
(1) An icELISA was developed with monoclonal antibody McAb6H73C9recognizing the plant growth regulator paclobutrazol (PBZ). The icELISA had a half-maximum inhibition concentration (IC50) and working range of approximately8.7and2.0～50.4ng/mL, respectively. Average recoveries of PBZ in the wheat (Triticum aestivum) kernel samples were between84.3and118.9%with relative standard deviations between3.9and14.2%. As determined by the icELISA and further confirmed by liquid chromatography-electrospray ionization quadrupole Orbitrap mass spectrometry (LC-ESI-MS) analysis, the maximum residue concentration was about0.07mg/kg in the kernel samples, which indicated that PBZ could transfer from PBZ-treated seedlings to the kernel samples. The correlation coefficient r2between icELISA and LC-ESI-MS results was0.979, which manifested that the developed icELISA was sensitive enough for monitoring PBZ residues in wheat kernels.
(2) The antigen of denatured Bt CrylAc protein was obtained by heat. The1.0mg/mL Bt CrylAc was dissolved in0.01M PBS, pH7.5and heated by100℃boiling for5min, which then was mixed with freund's complete adjuvant or freund's incomplete adjuvant, with the same volume. The hybridoma which could secrete antibody against native or heat denatured Bt CrylAc were screened by protein-coating ELISA and rabbit antiserum-protein-mouse antibody sandwich ELISA. Hybridomas1F2and3C9which could recognize coated native Bt Cry1Ac were obtained. And monoclonal hybridomas5E9C6、5E9D8、3E6A3、3E6A5and3E6E2which could recognize heat denature Bt Cry1Ac were developed. The monoclonal antibodies that secreted from5E9C6and3E6E2could apply to Western Blot. This study offers an experience for rapid development of mouse monoclonal antibody for Western Blot.
(3) The theory of small molecular compounds or proteins monoclonal antibody development alike each other, however the methods for target hybridoma screening were much different. Hybridoma which secrets antibody of small molecule compounds could be screened by icELISA method, based on the tilter and specificity of antibody that dissolved in nutrient solution. The research found that conformation of protein which adhere to solid phase may different from conformation of its' dissolved form. This could change the antigenic determinant. So hybridoma secrets antibody of active proteins should be screened by sandwich ELISA. And hybridoma which secrets antibody of denatured or for western blot usage could be developed by heat denatured antigen.
本研究制备了多效唑半抗原,并偶联蛋白制备得到完全抗原。制备了多效唑的单克隆抗体,并建立了多效唑间接竞争酶联免疫检测方法。研究发明了热变性Bt Cry1Ac蛋白免疫原的制备方法,通过免疫新西兰大耳兔及BALB/c小鼠分别制备了其多克隆抗血清和单克隆抗体,并建立热变性Bt CrylAc蛋白的双抗夹心免疫检测方法。
取得的研究结果和结论如下：
(1)制备得到多效唑的单克隆抗体McAb6H73C9株系,并建立了间接竞争酶联免疫检测方法(icELISA)。该icELISA的50%抑制浓度(IC50)值为8.7ng/g,标准曲线的工作范围为2.0-50.4ng/mL。在小麦籽粒中的添加回收率为84.3-118.9%,变异系数为3.9-14.2%。使用建立的icELISA检测实际样品发现最大的残留浓度为0.07mg/Kg,实验证明多效唑残留能够从喷施多效唑的幼苗转移到籽粒中,结果通过了液相色谱-电喷雾四级杆轨道阱质谱(LC-ESI-MS)验证,两种方法的相关系数0.979。这说明建立的icELISA的灵敏度足以检测小麦籽粒中的多效唑残留。
(2)首次成功制备了变性Bt CrylAc的免疫原。使用热变性的方式,将溶解在0.01M,pH=7.5的PBS中的1.0mg/mL Bt Cry1Ac蛋白在100℃C沸水浴处理5min,与弗氏完全佐剂或者不完全佐剂同体积混合乳化即为免疫原。分别使用包被蛋白ELISA和兔多抗-蛋白-鼠抗夹心ELISA的方式筛选得到了对非变性和热变性Bt Cry1Ac有阳性的杂交瘤。得到特异性识别包被的非变Bt Cry1Ac的杂交瘤株系为1F2和3C9,单克隆5E9C6、5E9D8、3E6A3、3E6A5和3E6E2分泌的抗体能特异性地识别热变性Bt CrylAc。5E9C6和3E6E2能够分泌专用于Western Blot的抗体。为今后快速制备免疫印迹用鼠单抗提供了技术经验。
(3)小分子化合物和大分子蛋白的单克隆抗体制备技术原理相同,但单克隆株系的筛选方法不同。小分子单克隆株系筛选主要通过间接竞争酶联免疫检测法检测细胞培养上清液中的阳性和抑制效果。研究发现,蛋白质吸附在固相载体上时空间构象与其在溶液中是不同的,这可能改变了抗原决定簇的构象。所以活性蛋白质的单克隆株系筛选应该通过双抗夹心的方法,变性蛋白和应用于免疫印迹抗体的筛选,可以通过热变性免疫原制备。
Paclobutrazol is a widely used plant growth regulator which could reduce the height of plants. The residue of paclobutrazol won't degradation for more than two years in the applied soil at room temperature. This research was to develop a sensitive and effective method for residue detection and monitor, and study the practical application. The safety was a much concern on transgenic proteins. Bacillus thuringiensis Bt CrylAc is one of the most commercial used insecticidal proteins in China. The common ELISA could not be used to detect heat denatured protein due to lack of antibody. This research has developed monoclonal antibody of heat denatured Bt Cry1Ac, which is a method basis for the development of practical enzyme linked immunosorbent assay (ELISA).
The hapten of paclobutrazol was synthesized. Complete antigen was obtained by conjugating hapten to BSA/OVA The indirect competitive enzyme linked immunosorbent assay (icELISA) for paclobutrazol was developed based on the obtained monoclonal antibody (McAb). The immunogen of heat denatured Bt CrylAc was invented. Rabbit antiserum and mouse monoclonal antibody were obtained by the immunization of New Zealand rabbit and BALB/c mouse, respectively. And the sandwich ELISA for heat denatured Bt CrylAc was developed
The main results as follows:
(1) An icELISA was developed with monoclonal antibody McAb6H73C9recognizing the plant growth regulator paclobutrazol (PBZ). The icELISA had a half-maximum inhibition concentration (IC50) and working range of approximately8.7and2.0～50.4ng/mL, respectively. Average recoveries of PBZ in the wheat (Triticum aestivum) kernel samples were between84.3and118.9%with relative standard deviations between3.9and14.2%. As determined by the icELISA and further confirmed by liquid chromatography-electrospray ionization quadrupole Orbitrap mass spectrometry (LC-ESI-MS) analysis, the maximum residue concentration was about0.07mg/kg in the kernel samples, which indicated that PBZ could transfer from PBZ-treated seedlings to the kernel samples. The correlation coefficient r2between icELISA and LC-ESI-MS results was0.979, which manifested that the developed icELISA was sensitive enough for monitoring PBZ residues in wheat kernels.
(2) The antigen of denatured Bt CrylAc protein was obtained by heat. The1.0mg/mL Bt CrylAc was dissolved in0.01M PBS, pH7.5and heated by100℃boiling for5min, which then was mixed with freund's complete adjuvant or freund's incomplete adjuvant, with the same volume. The hybridoma which could secrete antibody against native or heat denatured Bt CrylAc were screened by protein-coating ELISA and rabbit antiserum-protein-mouse antibody sandwich ELISA. Hybridomas1F2and3C9which could recognize coated native Bt Cry1Ac were obtained. And monoclonal hybridomas5E9C6、5E9D8、3E6A3、3E6A5and3E6E2which could recognize heat denature Bt Cry1Ac were developed. The monoclonal antibodies that secreted from5E9C6and3E6E2could apply to Western Blot. This study offers an experience for rapid development of mouse monoclonal antibody for Western Blot.
(3) The theory of small molecular compounds or proteins monoclonal antibody development alike each other, however the methods for target hybridoma screening were much different. Hybridoma which secrets antibody of small molecule compounds could be screened by icELISA method, based on the tilter and specificity of antibody that dissolved in nutrient solution. The research found that conformation of protein which adhere to solid phase may different from conformation of its' dissolved form. This could change the antigenic determinant. So hybridoma secrets antibody of active proteins should be screened by sandwich ELISA. And hybridoma which secrets antibody of denatured or for western blot usage could be developed by heat denatured antigen.
引文
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