应用CELL-SELEX筛选PANC-1细胞适配体技术研究
发布时间：2018-08-06 08:22
【摘要】：细胞适配体筛选可概括为前期准备、PCR扩增、次级文库制备和筛选四个部分,本文以这四个内容为导向,对CELL-SELEX技术筛选细胞适配体方法学进行了深入研究。本文首次以人胰腺癌细胞PANC-1为靶细胞进行适配体筛选研究。在对照细胞选择上引入了一种新的对照细胞选择策略,并确定了A549细胞为对照细胞的反向筛选。细胞状态稳定对适配体筛选非常重要,本文通过高传代频率的培养策略来维持细胞在整个筛选过程中状态稳定。结合软件与PCR扩增协同分析,对引物序列、PCR扩增效率和抗干扰性进行考察,确定了本文所用引物在细胞适配体筛选中的适用性。PCR在CELL-SELEX中被广范用于扩增次级文库。随机文库为模板的PCR难度大,不确定因素多。本文采用多种PCR方法对随机文库PCR特点进行考察。发现退火温度调节对扩增效率影响明显,对非特异性扩增作用较小;随机文库PCR对程序循环数极其敏感,循环数远少于常规PCR,对扩增效率和选择性影响较大。在次级文库PCR方法优化中,总结出一套具有广泛适用性的PCR优化策略,对细胞适配体筛选次级文库PCR具有指导意义。在次级文库制备中,针对单链分离过程建立了相应的监控办法,可对过程及时反馈,并确定待回收样品。通过延长PCR产物与Streptavidin-Agarose孵育时间,提高了分离柱对PCR产物的固载效率。借助核酸助沉剂(Acryl carrier),建立了适用于适配体筛选ssDNA乙醇低温沉淀回收方法,可以选择性回收目的ssDAN,回收率在80.0%以上。同时解决了ssDNA脱盐、PH平衡和ssDNA浓缩的问题。并建立了相应的定量表征方法。筛选是CELL-SELEX的主要内容,本文对细胞适配体筛选与筛选强度调整方法进行了研究。确定了5种基本筛选强度调整方式(细胞数量、ssDNA孵育浓度、孵育时间、孵育摇床转速和孵育后洗涤),发现5种调整方式之间的协同关系,确定了细胞量与文库孵育浓度在筛选强度调节中的基础地位,以及孵育后洗涤条件中洗涤时间在洗涤强度中的决定性作用。通过本次细胞适配体方法学研究成功搭建了PANC-1细胞适配体筛选平台,并初步筛得了PANC-1高亲和的候选适配体群。
[Abstract]:The selection of aptamer can be summarized as four parts: preparation for PCR amplification, preparation and screening of secondary library. In this paper, the methodology of screening aptamer by CELL-SELEX technology was studied. In this paper, the aptamer screening of human pancreatic cancer cell line PANC-1 was studied for the first time. A new control cell selection strategy was introduced and the reverse selection of A549 cells as control cells was determined. Cell state stability is very important for aptamer screening. Combined with software and PCR amplification synergistic analysis, the amplification efficiency and anti-interference ability of primer sequence were investigated, and the applicability of the primer in the screening of aptamer was determined. The PCR was widely used in CELL-SELEX to amplify secondary library. PCR with random library as template is difficult and uncertain. In this paper, various PCR methods are used to investigate the characteristics of random library PCR. It was found that annealing temperature had a significant effect on amplification efficiency and had little effect on non-specific amplification, and random library PCR was very sensitive to program cycle number, which was much less than conventional PCR, and had a great effect on amplification efficiency and selectivity. In the optimization of secondary library PCR method, a set of PCR optimization strategies with wide applicability is summarized, which is of guiding significance for the screening of secondary library PCR by cell aptamer. In the preparation of the secondary library, the corresponding monitoring method is established for the single chain separation process, which can provide timely feedback to the process and determine the samples to be recovered. By prolonging the incubation time of PCR products with Streptavidin-Agarose, the fixation efficiency of the separation column on PCR products was improved. A method of cryoprecipitation recovery of ssDNA ethanol was established by means of nucleic acid precipitator (Acryl carrier),. The recovery rate of ssDNA ethanol was over 80.0%, and the recovery rate was more than 80.0%. At the same time, the problems of PH equilibrium and ssDNA concentration of ssDNA desalting were solved. The corresponding quantitative characterization method was established. Screening is the main content of CELL-SELEX. In this paper, the selection and intensity adjustment of aptamer were studied. Five basic screening intensity adjustment methods (cell number and ssDNA incubation concentration, incubation time, incubating speed of shaking bed and washing after incubation) were determined, and the synergistic relationship between the five adjustment methods was found. The basic position of cell quantity and incubating concentration of library in the regulation of screening intensity and the decisive role of washing time in washing condition after incubation were determined. The PANC-1 aptamer screening platform was successfully constructed through the study of the aptamer methodology, and the candidate aptamer groups with high PANC-1 affinity were preliminarily screened.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.9
本文编号：2167117
[Abstract]:The selection of aptamer can be summarized as four parts: preparation for PCR amplification, preparation and screening of secondary library. In this paper, the methodology of screening aptamer by CELL-SELEX technology was studied. In this paper, the aptamer screening of human pancreatic cancer cell line PANC-1 was studied for the first time. A new control cell selection strategy was introduced and the reverse selection of A549 cells as control cells was determined. Cell state stability is very important for aptamer screening. Combined with software and PCR amplification synergistic analysis, the amplification efficiency and anti-interference ability of primer sequence were investigated, and the applicability of the primer in the screening of aptamer was determined. The PCR was widely used in CELL-SELEX to amplify secondary library. PCR with random library as template is difficult and uncertain. In this paper, various PCR methods are used to investigate the characteristics of random library PCR. It was found that annealing temperature had a significant effect on amplification efficiency and had little effect on non-specific amplification, and random library PCR was very sensitive to program cycle number, which was much less than conventional PCR, and had a great effect on amplification efficiency and selectivity. In the optimization of secondary library PCR method, a set of PCR optimization strategies with wide applicability is summarized, which is of guiding significance for the screening of secondary library PCR by cell aptamer. In the preparation of the secondary library, the corresponding monitoring method is established for the single chain separation process, which can provide timely feedback to the process and determine the samples to be recovered. By prolonging the incubation time of PCR products with Streptavidin-Agarose, the fixation efficiency of the separation column on PCR products was improved. A method of cryoprecipitation recovery of ssDNA ethanol was established by means of nucleic acid precipitator (Acryl carrier),. The recovery rate of ssDNA ethanol was over 80.0%, and the recovery rate was more than 80.0%. At the same time, the problems of PH equilibrium and ssDNA concentration of ssDNA desalting were solved. The corresponding quantitative characterization method was established. Screening is the main content of CELL-SELEX. In this paper, the selection and intensity adjustment of aptamer were studied. Five basic screening intensity adjustment methods (cell number and ssDNA incubation concentration, incubation time, incubating speed of shaking bed and washing after incubation) were determined, and the synergistic relationship between the five adjustment methods was found. The basic position of cell quantity and incubating concentration of library in the regulation of screening intensity and the decisive role of washing time in washing condition after incubation were determined. The PANC-1 aptamer screening platform was successfully constructed through the study of the aptamer methodology, and the candidate aptamer groups with high PANC-1 affinity were preliminarily screened.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.9
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