【作者】 李敏；

【导师】 万亚坤；

【作者基本信息】 东南大学 ， 遗传学， 2016， 硕士

【摘要】 人血清白蛋白是存在于人体内的一种重要而常见的蛋白质,是疾病检测与诊断的重要标记物之一。同时,关于如何去除血清中高丰度白蛋白以促进血浆蛋白质组学分析和生物标记物的发现,一直以来也备受人们的关注。因此,建立并发展一种有效而快速的白蛋白检测方法,具有重要意义。同时,关于如何建立有效去除血清高丰度蛋白质(白蛋白)的方法的问题也亟待解决,其对血清蛋白质组学的深入研究至关重要。本研究旨在对骆驼免疫人血清白蛋白、建立免疫文库的基础上,借助课题组成熟的噬菌体展示平台与技术,筛选并经PE-ELISA鉴定、测序、表达获得人血清白蛋白特异的单域抗体；再对这些单域抗体进行一系列特性分析并试图建立一套白蛋白检测的方法,同时试图利用NHS-活化珠子与单域抗体结合组装亲和柱、建立一套亲和去除的方法。经过努力,最终我们成功获得了一个高质量的噬菌体展示白蛋白单域抗体文库,并获得了四株特异针对人血清白蛋白的单域抗体。经过一系列分析,这些单域抗体表现出纯度好、产量高、特异性强、亲和力高等良好的特性。对于白蛋白的检测,我们成功建立了基于链霉亲和素-生物素固相反应系统的定向双单域抗体夹心检测的方法,线性范围10-1000 ng/mL、最低检测限达到21.2 ng/mL,为建立科学完善的白蛋白检测方法提供基础。最后,我们成功建立了一套基于单域抗体的亲和去除法,并成功地去除了血清中的高丰度蛋白(血清白蛋白),为血清蛋白质组学以及血清中疾病标志物的发现提供实验基础。更多还原

【Abstract】 Human serum albumin (HSA) plays role as vital biomarkers for disease-re la ted diagnosis, and its depletion approaches in plasma proteomic analysis and other biomarker discovery also have received much attention. Therefore, the importance of the methodological study of the HSA detection has been highlighted and effective depletion method for further study supposed to be developed.We constructed an immune phage display naobody library after immunization of a camels with HSA. By taking advantage of phage display technology, PE-ELISA, and sequencing, HSA-specific Nbs can be obtained. After expression and characterizing by a series of analysis, detection method would be developed. And for removal of HSA from serum, we would use these Nbs combined with NHS-Activated Sefinose Resin to assemble a spin tube filter.The immune phage display libraries were successfully constructed and four HSA-specific Nbs were obtained. After performing further characterization, these Nbs demonstrated good quality:high purity, good yield, strong specificity and high affinity. As for detection of HSA, the absorbance at 450 nm displayed a linearity with the concentrations of HSA ranging from 10 to 1000 ng·mL-1 and the minimum detection limit was calculated as 21.2 ng·mL-1. Finally, HSA in the blood serum sample was successfully depleted by means of nanobody-assembled resins.In conclusion, we successfully generated and characterized a number of nanobodies against HSA. And a directional, rapid and sensitive immunoassay for HSA detection was developed. Furthermore, an effective method for removal of HSA from serum have been established mainly based on Nbs and NHS-Activated Sefinose Resin. Our work indicated a promising future in further antigen detection and diagnostic applications.更多还原