應(yīng)用CELL-SELEX篩選PANC-1細(xì)胞適配體技術(shù)研究
發(fā)布時間：2018-08-06 08:22
【摘要】：細(xì)胞適配體篩選可概括為前期準(zhǔn)備、PCR擴增、次級文庫制備和篩選四個部分,本文以這四個內(nèi)容為導(dǎo)向,對CELL-SELEX技術(shù)篩選細(xì)胞適配體方法學(xué)進行了深入研究。本文首次以人胰腺癌細(xì)胞PANC-1為靶細(xì)胞進行適配體篩選研究。在對照細(xì)胞選擇上引入了一種新的對照細(xì)胞選擇策略,并確定了A549細(xì)胞為對照細(xì)胞的反向篩選。細(xì)胞狀態(tài)穩(wěn)定對適配體篩選非常重要,本文通過高傳代頻率的培養(yǎng)策略來維持細(xì)胞在整個篩選過程中狀態(tài)穩(wěn)定。結(jié)合軟件與PCR擴增協(xié)同分析,對引物序列、PCR擴增效率和抗干擾性進行考察,確定了本文所用引物在細(xì)胞適配體篩選中的適用性。PCR在CELL-SELEX中被廣范用于擴增次級文庫。隨機文庫為模板的PCR難度大,不確定因素多。本文采用多種PCR方法對隨機文庫PCR特點進行考察。發(fā)現(xiàn)退火溫度調(diào)節(jié)對擴增效率影響明顯,對非特異性擴增作用較小;隨機文庫PCR對程序循環(huán)數(shù)極其敏感,循環(huán)數(shù)遠少于常規(guī)PCR,對擴增效率和選擇性影響較大。在次級文庫PCR方法優(yōu)化中,總結(jié)出一套具有廣泛適用性的PCR優(yōu)化策略,對細(xì)胞適配體篩選次級文庫PCR具有指導(dǎo)意義。在次級文庫制備中,針對單鏈分離過程建立了相應(yīng)的監(jiān)控辦法,可對過程及時反饋,并確定待回收樣品。通過延長PCR產(chǎn)物與Streptavidin-Agarose孵育時間,提高了分離柱對PCR產(chǎn)物的固載效率。借助核酸助沉劑(Acryl carrier),建立了適用于適配體篩選ssDNA乙醇低溫沉淀回收方法,可以選擇性回收目的ssDAN,回收率在80.0%以上。同時解決了ssDNA脫鹽、PH平衡和ssDNA濃縮的問題。并建立了相應(yīng)的定量表征方法。篩選是CELL-SELEX的主要內(nèi)容,本文對細(xì)胞適配體篩選與篩選強度調(diào)整方法進行了研究。確定了5種基本篩選強度調(diào)整方式(細(xì)胞數(shù)量、ssDNA孵育濃度、孵育時間、孵育搖床轉(zhuǎn)速和孵育后洗滌),發(fā)現(xiàn)5種調(diào)整方式之間的協(xié)同關(guān)系,確定了細(xì)胞量與文庫孵育濃度在篩選強度調(diào)節(jié)中的基礎(chǔ)地位,以及孵育后洗滌條件中洗滌時間在洗滌強度中的決定性作用。通過本次細(xì)胞適配體方法學(xué)研究成功搭建了PANC-1細(xì)胞適配體篩選平臺,并初步篩得了PANC-1高親和的候選適配體群。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.9
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.9
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