共面多氯联苯抗体的制备及其用表面等离子共振方法检测的应用研究
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摘要
多氯联苯(Polychlorinated biphenyls,PCBs)是一类人工合成有机物,属于持久性有机污染物,对生态环境和人类构成潜在威胁,已成为全世界重点研究及监控的有机污染物之一。其中,共面多氯联苯(coplanar polychlorinated biphenyls,Co-PCBs),如PCB126因具有反应活性较高、三致作用强、危害严重等特点,对生态环境和人类健康影响最为显著,所以,本研究选择PCB126作为研究对象。
目前,相对于环境中PCBs的超痕量水平(一般为ng级或pg级),现有的PCBs分析方法(色谱法、生物法及免疫学法)对PCBs的检测限仅达到μg级水平,难以用于实际检测。因此,研究开发灵敏度高、可靠简便的PCBs分析检测方法意义重大。基于此,我们设计了以下研究方案:
第一步:将从文献中获得的抗Co-PCBs的抗体片段进行适当的构建后转入大肠杆菌M15中,表达生产Co-PCBs重组抗体;
第二步:利用分子自组装技术(Self-assembled Monolayer,SAM)和表面等离子共振技术(Surface Plasmon Resonance,SPR)对抗体特异性进行检测,同时检测重组抗体与PCB126的相互作用,并验证所得到的结果。
在本研究中,我们取得了以下成果:
1、通过基因工程的方法成功获得了Co-PCBs的抗体;
2、通过分子自组装的方法将Co-PCBs抗体成功地组装到金膜芯片上;
3、采用SPR技术对Co-PCBs抗体的特异性进行了检测,并检测到Co-PCBs抗体对PCB126检测限为5 10-4 pg/L;
4、利用SPR-2002对上述检测结果成功地进行了验证。
我们的实验成果将为环境中Co-PCBs的超痕量检测试剂盒的开发研制,提供一种快速、简单及可实时检测的检测手段。检测方法的建立将对全国各大水系中PCBs的时空动态分析、生态安全风险评价、污染治理及环境管理等提供重要依据。
Polychlorinated biphenyls (PCBs) are man-made compounds and persistent organic pollutants, and they have become one of the key research and monitoring organic pollutant in the word because they pose a potential threat to ecological environment and human. PCB126 which belongs Coplanar polychlorinated biphenyls (Co-PCBs), has the most significant effect on ecological environment and human health for its highly reactivity、strong carcinogenic、mutagenic and teratogenic effects、serious harm. So we chose PCB126 as the research object.
At present, relative to the ultra-trace levels of PCBs in environment (usually ng level or pg level), the existing PCBs analysis methods (chromatography, biological and immunological methods) can not be used for the actual test, because their detection limit for PCBs is onlyμg level. Therefore, the research and development of sensitive、reliable and easy detection of PCBs analysis method is of great significance.
Based these, we design the research program as follows:
The first step, the gene sequence of recombinant antibody against Co-PCBs indexed was constructed, and expressed it in Escherichia coli M15, then the recombinant antibody against Co-PCB was obtained by purifying and renaturation;
The second step, the Self-assembled Monolayer (SAM) and Surface Plasmon Resonance (SPR) were used to detect the specificity of antibody and the interaction between antibody and PCB126, and verify the results.
In these studies, we obtained the following results:
1、The antibody against Co-PCBs was obtained successfully through genetic engineering;
2、The antibody against Co-PCBs was assembled successfully on the surface of gold film through SAM;
3、The specificity of antibody was detected and the detection limit was about 5×10-4 pg/ L basing on SPR technology;
4、The above results was verified successfully on SPR-2002.
Our experimental results will provide a fast, simple and real-time detection methods for the development and research of Co-PCBs ultra-trace detection kit in the environment, and the establishment of detection methods will provide an important basis for the spatial analysis、ecological security risk assessment、pollution control and environmental management of PCBs in the major river systems of the country.
目前,相对于环境中PCBs的超痕量水平(一般为ng级或pg级),现有的PCBs分析方法(色谱法、生物法及免疫学法)对PCBs的检测限仅达到μg级水平,难以用于实际检测。因此,研究开发灵敏度高、可靠简便的PCBs分析检测方法意义重大。基于此,我们设计了以下研究方案:
第一步:将从文献中获得的抗Co-PCBs的抗体片段进行适当的构建后转入大肠杆菌M15中,表达生产Co-PCBs重组抗体;
第二步:利用分子自组装技术(Self-assembled Monolayer,SAM)和表面等离子共振技术(Surface Plasmon Resonance,SPR)对抗体特异性进行检测,同时检测重组抗体与PCB126的相互作用,并验证所得到的结果。
在本研究中,我们取得了以下成果:
1、通过基因工程的方法成功获得了Co-PCBs的抗体;
2、通过分子自组装的方法将Co-PCBs抗体成功地组装到金膜芯片上;
3、采用SPR技术对Co-PCBs抗体的特异性进行了检测,并检测到Co-PCBs抗体对PCB126检测限为5 10-4 pg/L;
4、利用SPR-2002对上述检测结果成功地进行了验证。
我们的实验成果将为环境中Co-PCBs的超痕量检测试剂盒的开发研制,提供一种快速、简单及可实时检测的检测手段。检测方法的建立将对全国各大水系中PCBs的时空动态分析、生态安全风险评价、污染治理及环境管理等提供重要依据。
Polychlorinated biphenyls (PCBs) are man-made compounds and persistent organic pollutants, and they have become one of the key research and monitoring organic pollutant in the word because they pose a potential threat to ecological environment and human. PCB126 which belongs Coplanar polychlorinated biphenyls (Co-PCBs), has the most significant effect on ecological environment and human health for its highly reactivity、strong carcinogenic、mutagenic and teratogenic effects、serious harm. So we chose PCB126 as the research object.
At present, relative to the ultra-trace levels of PCBs in environment (usually ng level or pg level), the existing PCBs analysis methods (chromatography, biological and immunological methods) can not be used for the actual test, because their detection limit for PCBs is onlyμg level. Therefore, the research and development of sensitive、reliable and easy detection of PCBs analysis method is of great significance.
Based these, we design the research program as follows:
The first step, the gene sequence of recombinant antibody against Co-PCBs indexed was constructed, and expressed it in Escherichia coli M15, then the recombinant antibody against Co-PCB was obtained by purifying and renaturation;
The second step, the Self-assembled Monolayer (SAM) and Surface Plasmon Resonance (SPR) were used to detect the specificity of antibody and the interaction between antibody and PCB126, and verify the results.
In these studies, we obtained the following results:
1、The antibody against Co-PCBs was obtained successfully through genetic engineering;
2、The antibody against Co-PCBs was assembled successfully on the surface of gold film through SAM;
3、The specificity of antibody was detected and the detection limit was about 5×10-4 pg/ L basing on SPR technology;
4、The above results was verified successfully on SPR-2002.
Our experimental results will provide a fast, simple and real-time detection methods for the development and research of Co-PCBs ultra-trace detection kit in the environment, and the establishment of detection methods will provide an important basis for the spatial analysis、ecological security risk assessment、pollution control and environmental management of PCBs in the major river systems of the country.
引文
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