噬菌体展示介导的免疫扩增病原检测方法研究
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摘要
当前感染性疾病的严峻现状和生物恐怖的潜在威胁使病原微生物的检测面临严重的挑战。本研究发展了一种新的病原检测方法，称为噬菌体展示介导的免疫扩增病原检测方法。该方法巧妙地利用了表面展示有单链抗体的重组噬菌体本身具有抗原结合分子和内部含有DNA的特点，通过表面展示的抗体识别目标抗原，利用内部的DNA作为核酸扩增的模板，把重组噬菌体作为载体直接介导完成免疫识别和信号检测过程。根据信号扩增方法的不同，可以分为噬菌体展示介导的免疫PCR和噬菌体展示介导的恒温扩增两种方法。噬菌体展示介导的免疫PCR是一种新型的免疫PCR方法，它利用重组噬菌体作为免疫PCR的载体。以汉坦病毒核蛋白为例，本研究论证了噬菌体展示介导的免疫PCR方法是一种高灵敏的病原检测方法，灵敏度比传统的酶联免疫吸附(ELISA)方法提高1，000-10，000倍，达到10 pg／ml。同时利用Real-time PCR技术结合标准曲线使该方法能够对抗原定量检测。检测结果表明重复性较好，在夹心检测的模式下2，000ng／ml到2 ng／ml的检测范围内R~2值达到0.96。本研究随后用恒温扩增方法代替PCR扩增噬菌体DNA发展噬菌体展示介导的恒温扩增方法，希望能摆脱对热循环仪的依赖。首先尝试噬菌体展示介导滚环扩增方法，该方法检测HIV P24抗原的灵敏度为100 pg／ml，但是所需的时间较长，而且检测的特异性不强。随后采用链置换扩增(strand displacement amplification，SDA)以取代滚环扩增作为信号检测的方法发展了噬菌体展示介导免疫链置换扩增方法。链置换扩增的效率很高，反应时间只需一个小时，检测HIV P24抗原和HBV HBsAg的灵敏度比传统的ELISA方法灵敏度提高1，000-10，000倍，同时结合荧光共振能量转移(fluorescence resonance energy transfer，FRET)探针做实时监测，使该方法的特异性大大增强。本研究发展的噬菌体展示介导的免疫扩增检测方法兼具免疫学检测方法的高特异性和核酸扩增方法的高灵敏度，而且成本低廉，有希望推广使用成为一种病原检测的常规方法。
The detection of pathogenic microbes is a great challenge because of the severesituation of infectious diseases and potential threaten of bioterrorism. A new detectionmethod for pathogen, called phage display mediated amplification method, wasdeveloped in this study. This method takes advantage of the recombinant phage,which displays antibody on thephage surface and includes DNA inside the phage.The surface displayed single chain variable fragment (scFv)and DNA inside thephage themselves can directly serve as detection antibody and nucleic acidamplification template, respectively. Thus, the recombinant phage, as a vector,mediates the process of immunological recognization and signal detection. Two kindsof methods, phage display mediated immuno-PCR and phage display mediatedisothermal amplification, were developed in this study based on the difference ofamplification method.Phage display mediated immuno-PCR (PD-IPCR)was a newtype of immuno-PCR method, in which the recombinant phage is a novel vector forimmuno-PCR. Hantaan virus nucleocapsid protein (NP) was selected as a model. Theresults demonstrated that the PD-IPCR method was high sensitive with a detectionlimit of 10 pg/ml for NP, which corresponded to an about 1,000-10,000-fold increasein sensitivity as compared to the enzyme-linked immunosorbent assays (ELISA)under analogous conditions. The real-time PCR technology was also applied in thismethod, which enabled the quantitative detection for antigen. The linear regressionwas good (R~2=0.96)for the signals of spiked samples against the logarithmicconcentrations from 2,000 ng/ml to 2 ng/ml in real-time sandwich PD-IPCR.Isothermal amplification methods were applied in the further study to develop phagedisplay mediated isothermal amplification methods for more broad applicationswithout relying on complicated machines. Rolling circle amplification (RCA) wasapplied first. The sensitivity of phage display mediated immuno-RCA for HIV P24antigen was 100 pg/ml. However, the amplification time of RCA is long and thespecificity is not high as other amplification methods. So, strand displacementamplification (SDA) was applied as the signal amplification to develop phage displaymediated immuno-SDA. The amplification efficiency of SDA reaction is so high thatthe reaction time is only 1h. The detection sensitivity for HIV P24 antigen and HBVHBsAg was increased 1,000 to 10,000 folds compared with conventional ELISA.FRET fluorescent probe was applied to develop real-time assay, which increase theassay specificity largely. The phage display mediated amplification methodsdeveloped in this study share main advantages of phage display and nucleic acid amplification technology. With the high sensitivity, specificity and low cost, thesemethods will be a routine technique in pathogen detection.
The detection of pathogenic microbes is a great challenge because of the severesituation of infectious diseases and potential threaten of bioterrorism. A new detectionmethod for pathogen, called phage display mediated amplification method, wasdeveloped in this study. This method takes advantage of the recombinant phage,which displays antibody on thephage surface and includes DNA inside the phage.The surface displayed single chain variable fragment (scFv)and DNA inside thephage themselves can directly serve as detection antibody and nucleic acidamplification template, respectively. Thus, the recombinant phage, as a vector,mediates the process of immunological recognization and signal detection. Two kindsof methods, phage display mediated immuno-PCR and phage display mediatedisothermal amplification, were developed in this study based on the difference ofamplification method.Phage display mediated immuno-PCR (PD-IPCR)was a newtype of immuno-PCR method, in which the recombinant phage is a novel vector forimmuno-PCR. Hantaan virus nucleocapsid protein (NP) was selected as a model. Theresults demonstrated that the PD-IPCR method was high sensitive with a detectionlimit of 10 pg/ml for NP, which corresponded to an about 1,000-10,000-fold increasein sensitivity as compared to the enzyme-linked immunosorbent assays (ELISA)under analogous conditions. The real-time PCR technology was also applied in thismethod, which enabled the quantitative detection for antigen. The linear regressionwas good (R~2=0.96)for the signals of spiked samples against the logarithmicconcentrations from 2,000 ng/ml to 2 ng/ml in real-time sandwich PD-IPCR.Isothermal amplification methods were applied in the further study to develop phagedisplay mediated isothermal amplification methods for more broad applicationswithout relying on complicated machines. Rolling circle amplification (RCA) wasapplied first. The sensitivity of phage display mediated immuno-RCA for HIV P24antigen was 100 pg/ml. However, the amplification time of RCA is long and thespecificity is not high as other amplification methods. So, strand displacementamplification (SDA) was applied as the signal amplification to develop phage displaymediated immuno-SDA. The amplification efficiency of SDA reaction is so high thatthe reaction time is only 1h. The detection sensitivity for HIV P24 antigen and HBVHBsAg was increased 1,000 to 10,000 folds compared with conventional ELISA.FRET fluorescent probe was applied to develop real-time assay, which increase theassay specificity largely. The phage display mediated amplification methodsdeveloped in this study share main advantages of phage display and nucleic acid amplification technology. With the high sensitivity, specificity and low cost, thesemethods will be a routine technique in pathogen detection.
引文
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