SPR生物传感器用于检测两种禽类病毒和转基因蛋白Cry1Ab的研究

发布时间：2018-04-28 06:06

本文选题：J亚群禽白血病病毒 + H9N2亚型禽流感病毒　；参考：《山东农业大学》2015年硕士论文

【摘要】：J亚群禽白血病病毒(ALV-J)和H9N2亚型禽流感病毒(H9N2 AIV)是两种严重危害世界各国包括我国的养禽业,并对其造成巨大经济损失的禽类病毒。目前这两种病毒的传统检测方法大都存在处理周期长、成本高,有时检测结果易出现假阳性等缺点。另外,转基因作物的大量种植造成了生态系统方面的担忧,转基因食品对人类的健康是否具有危害也存在较大争议。而当前现有的转基因蛋白的检测技术存在着操作复杂、耗时较长、成本高、重现性和准确性差的缺点。因此,本文将采用具有免标记样品、实时在线监测、灵敏快速检测的突出特点的表面等离子体共振(SPR)生物传感器检测这两种禽类病毒和转基因蛋白Cry1Ab。同时为了提高构建的SPR生物传感器的灵敏度,使检测结果更准确,Au/Ag复合纳米粒子和三角Ag纳米片作为增敏材料被引入传感器中。本文包括以下三部分实验内容。(1)实验构建了两种SPR生物传感器——传统模式生物传感器和Au/Ag复合纳米粒子增敏的生物传感器用于检测ALV-J。前者借助Au-S键使3-巯基丙酸(MPA)在SPR裸金膜芯片上形成一层密集的自组装单分子层,通过氨基与MPA中被活化剂活化后的羧基结合使蛋白A修饰到芯片上,接着芯片被乙醇胺灭活后,ALV-J抗体的非抗原结合位点与蛋白A结合使抗体修饰到芯片上,最后实现了对ALV-J抗原的检测;后者借助Au-S键使1,6-己二硫醇(HDT)一端在裸芯片上形成一层密集的自组装单分子层,另一端借助Au-S键、Ag-S键使Au/Ag复合纳米粒子修饰到芯片上,接下来传感器修饰各种物质与检测的方法同前者。结果两种传感器均能成功检测ALV-J抗原,检测限分别是1054 TCID50·mL 1和659 TCID50·mL 1,说明将Au/Ag复合纳米粒子引入SPR生物传感器中可有效提高传感器的灵敏度。而且进行的对比实验展现了制备的传感器具有良好的特异性。(2)实验构建了一种新型的SPR生物传感器用于检测H9N2 AIV。首先借助HDT将三角Ag纳米片修饰到芯片上,然后依次在芯片上修饰MPA、蛋白A和兔抗H9N2单克隆抗体,并最终实现对H9N2 AIV及裂解AIV的检测。结果传感器能顺利检测完整的H9N2 AIV,最低检测浓度为103 ELD50·mL 1。裂解病毒的浓度在50~103 ELD50·mL 1范围内与SPR信号成线性关系,检测限为28 ELD50·mL 1(S/N=3)。另外,此SPR生物传感器还展现了良好的检测特异性。(3)实验构建了两种SPR生物传感器(传感器1和传感器2)用于检测转基因蛋白Cry1Ab。在传感器2的芯片上依次修饰HDT、Au/Ag复合纳米粒子、MPA、蛋白A和鼠抗Cry1Ab单克隆抗体,从而实现对Cry1Ab蛋白的检测。传感器1与传感器2的构建过程相比,不同点在于没有先固定蛋白A而直接固定抗体。结果这两种传感器分别在Cry1Ab蛋白的浓度为10~500 ng·mL 1和8~1000 ng·mL 1时有良好的线性响应,检测限分别为5.0 ng·mL 1和4.8 ng·mL 1(S/N=3)。同时两种生物传感器均具有较高的特异性和较好的重现性。
[Abstract]:Subgroup J Avian Leukemia virus (ALV-JV) and H9N2 subtype Avian Influenza virus (H9N2 AIVA) are two kinds of avian viruses which seriously harm poultry industry in the world, including our country, and cause great economic losses to them. At present, most of the traditional detection methods of these two viruses have the disadvantages of long processing cycle, high cost and false positive results. In addition, the large number of genetically modified crops has caused ecosystem concerns, and whether GM food is harmful to human health is controversial. However, the existing detection techniques for transgenic proteins have the disadvantages of complex operation, long time consuming, high cost, poor reproducibility and accuracy. Therefore, a surface plasmon resonance (SPR) biosensor with the characteristics of non-labeled samples, real-time on-line monitoring and sensitive and rapid detection will be used to detect the two avian viruses and the transgenic protein Cry1Ab. At the same time, in order to improve the sensitivity of the constructed SPR biosensor and make the detection results more accurate, the Au/ Ag composite nanoparticles and triangular Ag nanoparticles were introduced into the sensor as sensitizing materials. In this paper, two kinds of SPR biosensors, traditional biosensors and Au/Ag composite nanoparticles biosensors were constructed to detect ALV-Js. The former formed a dense monolayer of self-assembly on SPR bare gold film chip by Au-S bond. Protein A was modified onto the chip by the binding of amino group to carboxyl group activated by activator in MPA. Then the chip was inactivated by ethanolamine and the non-antigen binding site of ALV-J antibody combined with protein A to modify the antibody onto the chip. Finally, the detection of ALV-J antigen was realized. The latter can form a dense self-assembled monolayer on the bare chip at one end and the Au/Ag composite nanoparticles on the chip by the Ag-S bond of the Au-S bond at one end of the chip by the aid of the Au-S bond, and the latter can form a dense self-assembled monolayer layer on the bare chip at one end and the other end on the other end by using the Ag-S bond of the Au-S bond. The sensor then modifies various substances and detects them in the same way as the former. Results the detection limits of ALV-J antigen were 1054 TCID50 / mL ~ (-1) and 659 TCID50 / mL ~ (-1), respectively, which indicated that the sensitivity of SPR biosensor could be improved effectively by introducing Au/Ag nanoparticles into SPR biosensor. Moreover, a new SPR biosensor was constructed for the detection of H9N2 SPR biosensor, which showed that the prepared biosensor has a good specificity. Firstly, the triangular Ag nanoparticles were modified onto the chip by HDT, then the MPA, protein A and rabbit anti- monoclonal antibody were modified on the chip in turn, and the detection of H9N2 AIV and AIV was finally realized. Results the sensor was able to detect the complete H9N2 AIV successfully, and the lowest detection concentration was 103 ELD50 / mL ~ (-1). The concentration of lytic virus was linearly correlated with SPR signal in the range of 50 ELD50 / 103 ELD50 / mL, and the detection limit was 28 ELD50 / mL ~ (-1) S / N ~ (-1) N ~ (3 +). In addition, two kinds of SPR biosensors (sensor 1 and sensor 2) were constructed to detect the transgenic protein Cry1Ab. The Cry1Ab protein was detected on the chip of sensor 2 by modifying MPA, protein A and mouse anti Cry1Ab monoclonal antibody in turn. The difference between sensor 1 and sensor 2 is that the antibody is not fixed first but directly. Results the two kinds of sensors showed good linear response at the concentration of 10 ~ 500ng / mL ~ (-1) of Cry1Ab protein and a concentration of 81000 ng / mL ~ (-1) of Cry1Ab protein, respectively. The detection limits were 5.0 ng / mL ~ (-1) and 4.8 ng / mL ~ (-1) ~ (-1) S / N ~ (-1) respectively. At the same time, the two biosensors have high specificity and good reproducibility.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65;TP212.3

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