基于模拟表位的玉米赤霉烯酮全抗原生物合成及其免疫学检测性能的研究
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摘要
真菌毒素(Mycotoxin)是一类由真菌产生的有毒次级代谢产物,大多显示有特定的器官毒性及致癌性,是食物链中常见的化学性污染物之一,也是目前影响我国食品安全的主要因素。对食品中的真菌毒素进行有效地监控和检测是防止其危害食品安全的重要手段。真菌毒素检测技术经过多年的研究,已经发展衍生出众多类型的检测模式,其中又以竞争型免疫检测体系应用最广。
建立小分子物质免疫学检测体系的前提和核心是全抗原的合成及抗体的制备。目前,小分子全抗原的制备主要依赖于化学合成的途径,然而该方法存在着合成步骤繁琐、反应时间长、副产物多、批次误差大等缺陷,特别是对于真菌毒素而言,其价格高昂且具致癌毒性,对操作人员及环境易造成毒害和污染,从而限制了免疫学检测方法的广泛使用。
本研究以常见的真菌毒素一玉米赤霉烯酮(Zearalenone, ZEN)为对象,通过噬菌体展示肽库、外源基因表达及分子定向改造技术,实现ZEN全抗原的生物合成,并将其应用于免疫学检测体系。主要研究内容为：1)ZEN噬菌体展示抗原模拟表位(十二肽)的淘选、鉴定及其结构研究；2)ZEN噬菌体展示抗原模拟表位免疫学检测性能的研究；3)ZEN新型模拟表位表达载体的构建、表达及其免疫学检测性能的研究；4)ZEN抗原模拟表位构效关系的初步研究,主要研究结果如下：
1淘选获得5种可特异性结合抗ZEN单抗(7G5)的ZEN噬菌体展示抗原模拟表位(十二肽),分别命名为Z5、Z8、Z14、Z15和Z18,采用ExPASy、 Swiss-pdbviewer软件分析其理论等电点、热稳定指数、消光系数、平均亲水性等物化性质并进行了二级结构的预测,结果表明5种模拟表位的两端均为无规则卷曲结构,中间为螺旋结构,理论等电点均小于7.0,对热稳定且具有亲水属性。
2建立了基于Z5、Z8、Z14、Z15、Z18的间接竞争Phage-ELISA标准曲线,其IC50值分别为23.5±2.0、2.6±0.4、7.0±0.8、1.2±0.3、14±1.6ng/mL。在此基础上,将ZEN抗原模拟表位进行了体外化学合成(多肽制备),并采用马来酰亚胺法将多肽与HRP酶偶联,建立了基于Z5-、Z8-、Z14-、Z15-、Z18-HRP偶联物的直接竞争ELISA标准曲线,其IC50值分别为15±2.4,2.0±0.6,7.2±1.5,0.7±0.1,11±3.1ng/mL。
3采用SPR研究了ZEN噬菌体展示抗原模拟表位与7G5的生物相互作用及其反应动力学,并进行了结合、解离曲线的分析以及平衡解离常数KD值的计算,结果显示Z5(十二肽)、P4(七肽)与7G5均呈现良好的线性结合关系,两者的反应动力学曲线相似,结合及解离点相近,其KD值分别为39.8,38.7nM, ZEN-BSA与7G5的KD值则为0.31nM。
4建立了基于ZEN噬菌体展示抗原模拟表位的固相膜免疫检测方法,该方法对于谷物样品中ZEN的Cut-off值为50μg/kg,检测时间10min,且可肉眼判定结果；同时构建了一种PVDF膜免疫快速检测装置,建立了可同时检测5种真菌毒素的PVDF膜免疫检测方法,该方法对于谷物样品中AFB1、ZEN、DON、 OTA、FB1的Cut-off值分别为20,60,1000,20,250μg/kg,检测时间10min且可肉眼判定结果。
5构建了Z5-, Z8-, Z14-, Z15-, Z18-pMal-PIII和Z15-pRX-AP表达载体,经IPTG诱导后,分别获得了Z5-, Z8-, Z14-, Z15-, Z18-MBP和Z15-AP融合蛋白,建立的间接竞争ELISA标准曲线显示均呈现典型的竞争抑制关系,其IC5o值分别为1.5±0.2,1.6±0.3,3.5±0.5,7.2±0.8,1.8±0.4ng/mL。Z15-AP融合蛋白与AP底物显色液可显色,但与ZEN抗体的结合能力较弱,OD值在0.3-0.5之间。
6研究了ZEN抗原模拟表位的串联拷贝数、间隔臂及环化结构与其免疫学检测性能的关系,结果显示串联拷贝数、间隔臂并不是影响ZEN模拟表位免疫学检测性能的关键因素；相比于线性结构,ZEN模拟表位的环化结构在作为竞争抗原应用于直接竞争ELISA体系时,其检测灵敏度提高了近60%。建立了基于Z5-MBP融合蛋白的间接竞争ELISA方法检测谷物中的ZEN,与商业化的ZENELISA检测试剂盒(德国Biopharm公司)进行检测比对,结果显示两者之间无显著性的差异,表明ZEN新型模拟表位可实现ZEN全抗原的替代,并应用于免疫学检测体系。
Mycotoxins are toxic secondary metabolites of fungal origin; most of mycotoxins are hazardous to the health of both humans and animals and now recognised as a major cause of food safety issue in China. Detection and control of mycotoxins contamination play an important role in food safety system. Currently, many mycotoxin detection methods have been developed, the competitive immunoassay is now the most applied and representative method for the rapid screening analysis of mycotoxins.
The preparation of antibody and complete antigen is the precondition and core of setting up competitive immunoassay for small analytes. However, at present, the complete antigen preparation of small analytes including mycotoxins is mainly dependent on the chemical synthesis approach, while the process of chemical synthesis is time-consuming, costly and might be toxic to manufacturers and users, which often hinders its extensive application.
In this study, we combine the technology of phage displayed peptide library, molecular simulation and exogenous gene expression to develop the method of biosynthesis of complete antigen for model small molecules mycotoxin, zearalenone(ZEN) and achieve its application in immunoassay. The main contents of research are:(i) the biopanning of ZEN mimotope by phage displayed peptide library (12-mer) and its identification;(ii) the research of the immunological detection performance of ZEN mimotope;(iii) construction and expression of ZEN mimotope-based vectors and the research of immunological detection performance of expression fusion proteins;(iv) elucidating the structure-activity relationship of the ZEN mimotopes. The main research results are as follows:
1. Monoclonal antibody (7G5), which recognizes the ZEN, was used to select for peptides that mimic ZEN by employing a library of filamentous phages that have12-mer peptides on their surfaces. After three rounds of biopanning, five mimotope peptides were obtained to be able to mimic ZEN in binding with the7G5and designated Z5, Z8, Z14, Z15, and Z18. The theoretical isoelectric point, heat stability index, extinction coefficient, average hydrophilic physicochemical properties and secondary structure of ZEN mimotope were analyzed by ExPasy and Swiss-pdbviewer softwares. The results showed that five mimotope have the same secondary structure, which were predominantly in radom coil and helix, and the same hydrophilic, heat stable properties.
2. The inhibition curves of ZEN by indirect competitive ELISA were established with ZEN mimotopes, the IC50value was23.5±2.0、2.6±0.4、7.0±0.8、1.2±0.3、14±1.6ng/mL for Z5, Z8, Z14, Z15, and Z18, respectively. Furthermore, the peptides which have the same amino acids as ZEN mimotope were synthesized and conjugated with HRP (Z5-, Z8-, Z14-, Z15-, and Z18-HRP) by maleimide method; the inhibition curves of ZEN by direct competitive ELISA were established with peptide-HRP conjugates, and the IC50value was15±2.4,2.0±0.6,7.2±1.5,0.7±0.1,11±3.1ng/mL for Z5-, Z8-, Z14-, Z15-, and Z18-HRP, respectively.
3. The Z5(12-mer), P4(7-mer), and ZEN-BSA conjugates were immobilized on sensor chip, the reaction kinetics between mimotope/conjugates and7G5were detected by surface plasmon resonance (SPR) analysis. The results showed that sensorgram curves obtained were well-behaved and the SPR response unit (RU) caused by7G5binding increased as the concentration of antibody increased, which is in accordance with the indirect immunoassay principle. The equilibrium dissociation constant (KD) measured for P4:7G5(KD=38.7nM) and Z5:7G5(KD=39.8nM) was-100-fold higher than that measured for ZEN-BSA:7G5(KD=0.31nM).
4. A polyvinylidene fluoride (PVDF) membrane-based test device was constructed to develop a non-instrumental assay performed with ZEN mimotope. The cut-off level for this method of detecting ZEN, assessed visually, was50μg kg-1and the final results can be obtained within10min. Furthermore, a PVDF membrane-based dot immunoassay for rapid and simultaneous detection of multi-mycotoxins in cereal samples is also developed. The cut-off level for this method, assessed visually, were20,60,1000,20, and250μg kg-1for aflatoxin B1, zearalenone, deoxynivalenol, ochratoxin A, and fumonisins B1respectively, the final results can be obtained within10min.
5. Five ZEN mimotope-pMal-pⅢ expression vector (Z5-, Z5-, Z8-, Z14-, Z15-, and Z18-pMal-pⅢ) were constructed to express mimotope-MBP fusion proteins which mime ZEN complete antigen. The mimotope-MBP fusion proteins were coated in microplates, and the inhibition curves of ZEN by indirect competitive ELISA were established, the IC50value was1.5±0.2,1.6±0.3,3.5±0.5,7.2±0.8,1.8±0.4ng/mL for Z5-, Z5-, Z8-, Z14-, Z15-MBP fusion protein coated microplates, respectively. Z15-pRX-AP expresssion vector was also constructed to express Z15-AP fusion proteins, whilie the binding ability between Z15-AP and7G5is very low.
6. The structure-activity relationship of the ZEN mimotopes was studied, the results showed that the linker peptides and multi-copies structure were not key factors which effect the immunological detection performance of ZEN mimotope; while the cyclized Z15based direct immunoassay is more sensitive than Z15based.
建立小分子物质免疫学检测体系的前提和核心是全抗原的合成及抗体的制备。目前,小分子全抗原的制备主要依赖于化学合成的途径,然而该方法存在着合成步骤繁琐、反应时间长、副产物多、批次误差大等缺陷,特别是对于真菌毒素而言,其价格高昂且具致癌毒性,对操作人员及环境易造成毒害和污染,从而限制了免疫学检测方法的广泛使用。
本研究以常见的真菌毒素一玉米赤霉烯酮(Zearalenone, ZEN)为对象,通过噬菌体展示肽库、外源基因表达及分子定向改造技术,实现ZEN全抗原的生物合成,并将其应用于免疫学检测体系。主要研究内容为：1)ZEN噬菌体展示抗原模拟表位(十二肽)的淘选、鉴定及其结构研究；2)ZEN噬菌体展示抗原模拟表位免疫学检测性能的研究；3)ZEN新型模拟表位表达载体的构建、表达及其免疫学检测性能的研究；4)ZEN抗原模拟表位构效关系的初步研究,主要研究结果如下：
1淘选获得5种可特异性结合抗ZEN单抗(7G5)的ZEN噬菌体展示抗原模拟表位(十二肽),分别命名为Z5、Z8、Z14、Z15和Z18,采用ExPASy、 Swiss-pdbviewer软件分析其理论等电点、热稳定指数、消光系数、平均亲水性等物化性质并进行了二级结构的预测,结果表明5种模拟表位的两端均为无规则卷曲结构,中间为螺旋结构,理论等电点均小于7.0,对热稳定且具有亲水属性。
2建立了基于Z5、Z8、Z14、Z15、Z18的间接竞争Phage-ELISA标准曲线,其IC50值分别为23.5±2.0、2.6±0.4、7.0±0.8、1.2±0.3、14±1.6ng/mL。在此基础上,将ZEN抗原模拟表位进行了体外化学合成(多肽制备),并采用马来酰亚胺法将多肽与HRP酶偶联,建立了基于Z5-、Z8-、Z14-、Z15-、Z18-HRP偶联物的直接竞争ELISA标准曲线,其IC50值分别为15±2.4,2.0±0.6,7.2±1.5,0.7±0.1,11±3.1ng/mL。
3采用SPR研究了ZEN噬菌体展示抗原模拟表位与7G5的生物相互作用及其反应动力学,并进行了结合、解离曲线的分析以及平衡解离常数KD值的计算,结果显示Z5(十二肽)、P4(七肽)与7G5均呈现良好的线性结合关系,两者的反应动力学曲线相似,结合及解离点相近,其KD值分别为39.8,38.7nM, ZEN-BSA与7G5的KD值则为0.31nM。
4建立了基于ZEN噬菌体展示抗原模拟表位的固相膜免疫检测方法,该方法对于谷物样品中ZEN的Cut-off值为50μg/kg,检测时间10min,且可肉眼判定结果；同时构建了一种PVDF膜免疫快速检测装置,建立了可同时检测5种真菌毒素的PVDF膜免疫检测方法,该方法对于谷物样品中AFB1、ZEN、DON、 OTA、FB1的Cut-off值分别为20,60,1000,20,250μg/kg,检测时间10min且可肉眼判定结果。
5构建了Z5-, Z8-, Z14-, Z15-, Z18-pMal-PIII和Z15-pRX-AP表达载体,经IPTG诱导后,分别获得了Z5-, Z8-, Z14-, Z15-, Z18-MBP和Z15-AP融合蛋白,建立的间接竞争ELISA标准曲线显示均呈现典型的竞争抑制关系,其IC5o值分别为1.5±0.2,1.6±0.3,3.5±0.5,7.2±0.8,1.8±0.4ng/mL。Z15-AP融合蛋白与AP底物显色液可显色,但与ZEN抗体的结合能力较弱,OD值在0.3-0.5之间。
6研究了ZEN抗原模拟表位的串联拷贝数、间隔臂及环化结构与其免疫学检测性能的关系,结果显示串联拷贝数、间隔臂并不是影响ZEN模拟表位免疫学检测性能的关键因素；相比于线性结构,ZEN模拟表位的环化结构在作为竞争抗原应用于直接竞争ELISA体系时,其检测灵敏度提高了近60%。建立了基于Z5-MBP融合蛋白的间接竞争ELISA方法检测谷物中的ZEN,与商业化的ZENELISA检测试剂盒(德国Biopharm公司)进行检测比对,结果显示两者之间无显著性的差异,表明ZEN新型模拟表位可实现ZEN全抗原的替代,并应用于免疫学检测体系。
Mycotoxins are toxic secondary metabolites of fungal origin; most of mycotoxins are hazardous to the health of both humans and animals and now recognised as a major cause of food safety issue in China. Detection and control of mycotoxins contamination play an important role in food safety system. Currently, many mycotoxin detection methods have been developed, the competitive immunoassay is now the most applied and representative method for the rapid screening analysis of mycotoxins.
The preparation of antibody and complete antigen is the precondition and core of setting up competitive immunoassay for small analytes. However, at present, the complete antigen preparation of small analytes including mycotoxins is mainly dependent on the chemical synthesis approach, while the process of chemical synthesis is time-consuming, costly and might be toxic to manufacturers and users, which often hinders its extensive application.
In this study, we combine the technology of phage displayed peptide library, molecular simulation and exogenous gene expression to develop the method of biosynthesis of complete antigen for model small molecules mycotoxin, zearalenone(ZEN) and achieve its application in immunoassay. The main contents of research are:(i) the biopanning of ZEN mimotope by phage displayed peptide library (12-mer) and its identification;(ii) the research of the immunological detection performance of ZEN mimotope;(iii) construction and expression of ZEN mimotope-based vectors and the research of immunological detection performance of expression fusion proteins;(iv) elucidating the structure-activity relationship of the ZEN mimotopes. The main research results are as follows:
1. Monoclonal antibody (7G5), which recognizes the ZEN, was used to select for peptides that mimic ZEN by employing a library of filamentous phages that have12-mer peptides on their surfaces. After three rounds of biopanning, five mimotope peptides were obtained to be able to mimic ZEN in binding with the7G5and designated Z5, Z8, Z14, Z15, and Z18. The theoretical isoelectric point, heat stability index, extinction coefficient, average hydrophilic physicochemical properties and secondary structure of ZEN mimotope were analyzed by ExPasy and Swiss-pdbviewer softwares. The results showed that five mimotope have the same secondary structure, which were predominantly in radom coil and helix, and the same hydrophilic, heat stable properties.
2. The inhibition curves of ZEN by indirect competitive ELISA were established with ZEN mimotopes, the IC50value was23.5±2.0、2.6±0.4、7.0±0.8、1.2±0.3、14±1.6ng/mL for Z5, Z8, Z14, Z15, and Z18, respectively. Furthermore, the peptides which have the same amino acids as ZEN mimotope were synthesized and conjugated with HRP (Z5-, Z8-, Z14-, Z15-, and Z18-HRP) by maleimide method; the inhibition curves of ZEN by direct competitive ELISA were established with peptide-HRP conjugates, and the IC50value was15±2.4,2.0±0.6,7.2±1.5,0.7±0.1,11±3.1ng/mL for Z5-, Z8-, Z14-, Z15-, and Z18-HRP, respectively.
3. The Z5(12-mer), P4(7-mer), and ZEN-BSA conjugates were immobilized on sensor chip, the reaction kinetics between mimotope/conjugates and7G5were detected by surface plasmon resonance (SPR) analysis. The results showed that sensorgram curves obtained were well-behaved and the SPR response unit (RU) caused by7G5binding increased as the concentration of antibody increased, which is in accordance with the indirect immunoassay principle. The equilibrium dissociation constant (KD) measured for P4:7G5(KD=38.7nM) and Z5:7G5(KD=39.8nM) was-100-fold higher than that measured for ZEN-BSA:7G5(KD=0.31nM).
4. A polyvinylidene fluoride (PVDF) membrane-based test device was constructed to develop a non-instrumental assay performed with ZEN mimotope. The cut-off level for this method of detecting ZEN, assessed visually, was50μg kg-1and the final results can be obtained within10min. Furthermore, a PVDF membrane-based dot immunoassay for rapid and simultaneous detection of multi-mycotoxins in cereal samples is also developed. The cut-off level for this method, assessed visually, were20,60,1000,20, and250μg kg-1for aflatoxin B1, zearalenone, deoxynivalenol, ochratoxin A, and fumonisins B1respectively, the final results can be obtained within10min.
5. Five ZEN mimotope-pMal-pⅢ expression vector (Z5-, Z5-, Z8-, Z14-, Z15-, and Z18-pMal-pⅢ) were constructed to express mimotope-MBP fusion proteins which mime ZEN complete antigen. The mimotope-MBP fusion proteins were coated in microplates, and the inhibition curves of ZEN by indirect competitive ELISA were established, the IC50value was1.5±0.2,1.6±0.3,3.5±0.5,7.2±0.8,1.8±0.4ng/mL for Z5-, Z5-, Z8-, Z14-, Z15-MBP fusion protein coated microplates, respectively. Z15-pRX-AP expresssion vector was also constructed to express Z15-AP fusion proteins, whilie the binding ability between Z15-AP and7G5is very low.
6. The structure-activity relationship of the ZEN mimotopes was studied, the results showed that the linker peptides and multi-copies structure were not key factors which effect the immunological detection performance of ZEN mimotope; while the cyclized Z15based direct immunoassay is more sensitive than Z15based.
引文
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