本文關(guān)鍵詞：體外篩選銅綠假單胞菌適體的研究及初步應(yīng)用　出處：《福建醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 銅綠假單胞菌 適體 SELEX 反篩 熒光基團(tuán) dsDNA ssDNA PCR

【摘要】： 【目的】 體外篩選出高親和力高特異性針對銅綠假單胞菌的適體群;測定適體與銅綠假單胞菌的結(jié)合活性,確定其結(jié)合率及特異性。 【方法】 1.在體外構(gòu)建兩端為固定序列,中間為35個堿基隨機(jī)區(qū),全長78個堿基的寡核苷酸庫,以純培養(yǎng)的銅綠假單胞菌為靶標(biāo),將二者混合,經(jīng)過九輪的吸附-漂洗-洗脫-擴(kuò)增的SELEX過程,篩選針對銅綠假單胞菌高親和力高特異性的適體,并從第五輪起每隔一輪分別用熒光假單胞、斯氏假單胞和產(chǎn)堿假單胞菌進(jìn)行反篩,以提高篩選的特異性。每隔一輪利用地高辛-抗地高辛抗體-堿性磷酸酶系統(tǒng)測定ssDNA富集庫與銅綠假單胞菌的結(jié)合率。 2.將第九輪的篩選產(chǎn)物——寡核苷酸片斷進(jìn)行克隆、測序并用相關(guān)軟件分析其一級和二級結(jié)構(gòu)。 3.用微量熒光分光光度計檢測熒光素標(biāo)記適體與銅綠假單胞菌的結(jié)合率,在5個測序克隆中選出結(jié)合率最高的一條適體,分別與銅綠假單胞、門多薩假單胞、斯氏假單胞和產(chǎn)堿假單胞菌做結(jié)合反應(yīng),以初步檢測其特異性。 【結(jié)果】 1.經(jīng)過九輪的篩選和擴(kuò)增,使得針對銅綠假單胞菌的適體逐步得到富集,每輪的ssDNA文庫與銅綠假單胞菌的結(jié)合率呈上升趨勢。將每輪的篩選產(chǎn)物用標(biāo)有地高辛的引物擴(kuò)增后,與銅綠假單胞菌進(jìn)行結(jié)合實(shí)驗(yàn),地高辛-抗地高辛抗體-堿性磷酸酶顯色系統(tǒng)提示,第九輪篩選獲得的富集庫與銅綠假單胞菌結(jié)合后顯色的OD值較第一輪的OD值提高將近3.2倍。 2.在200個克隆子中隨機(jī)選取27個克隆進(jìn)行了測序,將測序結(jié)果按堿基多少分成A、B兩群,A群19條與預(yù)期片段大小一致;B群為中間隨機(jī)區(qū)少于35個堿基的片段。將測序適體以計算機(jī)軟件分析其保守序列及二級結(jié)構(gòu)。二級結(jié)構(gòu)預(yù)測以莖-環(huán)結(jié)構(gòu)為主,這可能是適體與靶標(biāo)作用的結(jié)構(gòu)基礎(chǔ)。其中適體序列完全相同的共有6條,此序列隨機(jī)區(qū)富含20個G,可能為G四聚體的形成基礎(chǔ)。此外,有些序列是單個序列,與任何家族無同源。某些一級結(jié)構(gòu)無同源性的適體克隆二級結(jié)構(gòu)模擬卻極為相似,如2-26、2-61、2-119號克隆,2-24、2-46號克隆,2-12、2-135克隆序列等。 3.結(jié)合實(shí)驗(yàn)顯示,2-61號克隆適體與銅綠假單胞菌的結(jié)合率最高,為15.3%,2-106號克隆適體與銅綠假單胞菌的結(jié)合率最低,為5.6%;2-61號克隆適體與銅綠假單胞、斯氏假單胞菌、產(chǎn)堿假單胞菌和門多薩假單胞,進(jìn)行結(jié)合反應(yīng)后,測得F值分別為16.6%、3.8%、2.6%和4.5%。 【結(jié)論】 本研究在國內(nèi)首次利用反向-指數(shù)富集配體系統(tǒng)進(jìn)化(SELEX)技術(shù)篩選出針對銅綠假單胞菌的ssDNA適體,并對篩選出的適體進(jìn)行了結(jié)合率及特異性的檢測,初步確定了結(jié)合率較高特異性較好的適體分子,為熒光基團(tuán)標(biāo)記的適體快速鑒定銅綠假單胞菌檢測方法的建立奠定了基礎(chǔ)。
[Abstract]:[purpose] In vitro, the aptamer groups with high affinity and specificity against Pseudomonas aeruginosa were screened, and the binding activity between aptamer and Pseudomonas aeruginosa was determined, and the binding rate and specificity of the aptamer and Pseudomonas aeruginosa were determined. [methods] 1. A 78 base oligonucleotide library with a fixed sequence at both ends and 35 base random regions in the middle was constructed in vitro. The two oligonucleotides were mixed with pure cultured Pseudomonas aeruginosa as the target. After nine rounds of SELEX process of adsorption, rinsing, elution and amplification, the aptamers with high affinity and high specificity for Pseudomonas aeruginosa were screened, and fluorescent pseudomonas were used every other round from 5th rounds. Pseudomonas sp. And Pseudomonas sp. In order to improve the specificity of screening, the binding rate of ssDNA enrichment library with Pseudomonas aeruginosa was determined by using digoxigenin-anti-digoxin antibody alkaline phosphatase system every other round. 2. The oligonucleotide fragment of 9th rounds of screening product was cloned and sequenced and its primary and secondary structures were analyzed by software. 3. The binding rate of fluorescein labeled aptamer to Pseudomonas aeruginosa was detected by microfluorimetric spectrophotometer. One aptamer with the highest binding rate was selected among the five sequencing clones, and the aptamer was associated with the Pseudomonas aeruginosa (Pseudomonas aeruginosa). Mendoza pseudomonas, Stemont's pseudomonas and Pseudomonas alcaligenes were combined to detect the specificity of Mendoza pseudomonas. [results] 1. After nine rounds of screening and amplification, the aptamer for Pseudomonas aeruginosa was gradually enriched. The binding rate of each ssDNA library to Pseudomonas aeruginosa was on the rise. The screening products were amplified with digoxigenin primers and then combined with Pseudomonas aeruginosa. The results of digoxigenin-anti-digoxin antibody alkaline phosphatase system suggested that the OD value of the enrichment library combined with Pseudomonas aeruginosa was 3.2 times higher than that of the first round. 2. Among the 200 clones, 27 clones were randomly selected and sequenced. According to the number of bases, the sequencing results were divided into two groups of Agna B and A groups, 19 of which were consistent with the expected size of the fragments. Group B was a fragment with less than 35 bases in the middle random region. The sequenced aptamer was analyzed by computer software for its conserved sequence and secondary structure. The secondary structure was predicted mainly by stem-loop structure. This may be the structural basis of the interaction between aptamer and target. There are 6 aptamers with identical aptamer sequences, and the random region of the aptamer sequence is rich in 20 Gs, which may be the basis for the formation of G tetramer. Some sequences are single sequences and have no homology with any family. The secondary structure simulation of some aptamer clones with no homology in some primary structures is very similar, such as clone 2-262-61C2-119. Cloning sequence of 2-24N 2-46, 2-12, 2-135, et al. 3. The binding rate between clone aptamer No. 2-61 and Pseudomonas aeruginosa was the highest, and the binding rate between clone aptamer No. 2-106 and Pseudomonas aeruginosa was the lowest (5.6B). After the binding reaction of aptamer No. 2-61 with Pseudomonas aeruginosa, Pseudomonas sp., Pseudomonas sp. And Pseudomonas monocytogenes Mendoza, the F values were 16. 6% and 3. 8%, respectively. 2.6% and 4.5. [conclusion] In this study, ssDNA aptamers against Pseudomonas aeruginosa were screened by reverse exponential enrichment ligand phylogeny (SELEX) technique for the first time in China. The binding rate and specificity of the selected aptamers were determined, and the aptamer molecules with higher binding rate and better specificity were preliminarily determined. The results laid a foundation for rapid identification of Pseudomonas aeruginosa by fluorescent group labeling.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R378

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