體外SELEX技術(shù)篩選滅活銅綠假單胞菌適體的研究及初步應(yīng)用
發(fā)布時(shí)間：2018-08-08 11:05
【摘要】： 【目的】 利用指數(shù)富集配基的系統(tǒng)進(jìn)化(SELEX)技術(shù),以滅活的銅綠假單胞菌作為復(fù)合靶分子,從體外合成的96個(gè)nt的隨機(jī)寡核苷酸文庫(kù)中篩選出與銅綠假單胞菌高親和力高特異性的適體,并建立熒光基團(tuán)標(biāo)記適體快速鑒定銅綠假單胞菌的檢測(cè)方法。 【方法】 1.在體外構(gòu)建兩端為固定序列,中間為60個(gè)nt的隨機(jī)序列,全長(zhǎng)96個(gè)nt的寡核苷酸文庫(kù),以滅活的銅綠假單胞菌為靶標(biāo),經(jīng)SELEX篩選,篩選出針對(duì)銅綠假單胞菌高親和力高特異性的適體,用酶連接適體直接檢測(cè)法(Enzyme-linked aptamer direct assay,ELADA)利用地高辛-抗地高辛抗體-堿性磷酸酶系統(tǒng)進(jìn)行ssDNA富集庫(kù)與銅綠假單胞菌的結(jié)合測(cè)定。 2.我們?cè)趯?shí)驗(yàn)中從第12輪和第14輪分別同時(shí)進(jìn)行ssDNA文庫(kù)與嗜麥芽窄食單胞菌和鮑曼不動(dòng)桿菌反篩和未反篩的兩條思路,運(yùn)用ELADA間接比較反篩和未反篩獲得的ssDNA富集庫(kù)與銅綠假單胞菌的結(jié)合力。 3.將第6輪、第10輪和第15輪篩選產(chǎn)物分別進(jìn)行擴(kuò)增、純化, TA克隆、測(cè)序并用相關(guān)軟件分析其一級(jí)和二級(jí)結(jié)構(gòu)。 4.用ELADA進(jìn)行克隆適體與銅綠假單胞菌的結(jié)合測(cè)定,選出OD值明顯高的3個(gè)克隆適體,并對(duì)其進(jìn)行親和力測(cè)定;然后對(duì)親和力最高的克隆適體,進(jìn)一步進(jìn)行靈敏度和特異性分析。運(yùn)用DFA(dot filtration assay, DFA)對(duì)F23克隆適體與非發(fā)酵菌進(jìn)行特異性檢測(cè)。 5.建立熒光基團(tuán)標(biāo)記適體快速鑒定銅綠假單胞菌檢測(cè)方法:將F23適體進(jìn)行5’-FAM和5’-TAMRA熒光基團(tuán)標(biāo)記直接和非發(fā)酵菌結(jié)合、洗滌、封片,熒光顯微鏡檢查。 【結(jié)果】 1.隨著篩選輪數(shù)的增加,銅綠假單胞菌與ssDNA富集庫(kù)結(jié)合的OD值逐漸增加,第十二輪反篩選獲得的富集庫(kù)與銅綠假單胞菌結(jié)合后顯色的OD值(0.448)是第一輪OD值(0.022)的23倍。當(dāng)ssDNA富集庫(kù)與銅綠假單胞菌上的結(jié)合位點(diǎn)達(dá)到一個(gè)飽和狀態(tài)時(shí),吸附到銅綠假單胞菌上的ssDNA就不再增加。 2.經(jīng)過反篩和未反篩的ssDNA富集庫(kù)與銅綠假單胞菌結(jié)合的OD值有顯著差異,從最低的第15輪的反篩和未反篩的OD比值為1.3倍,到最高的第14輪比值的53倍。 3.克隆序列結(jié)果發(fā)現(xiàn)第6輪、第10輪和第15輪未反篩的序列均無富集,而第15輪反篩的克隆子進(jìn)行測(cè)序,發(fā)現(xiàn)24個(gè)預(yù)期隨機(jī)序列中有2條序列富集,幾乎完全一致(F23和F47),同源性達(dá)97%,利用ClustalX和Mega2軟件分析序列的一級(jí)結(jié)構(gòu)的同源性和分類,分成10個(gè)家族(Family),利用Mfold sever軟件模擬二級(jí)結(jié)構(gòu)。 4.克隆適體F17、F23和F47與銅綠假單胞菌結(jié)合的OD值明顯增高;進(jìn)行親和力測(cè)定,可知:F23的Kd為14.55nM,F47為77.46nM,F17為493.3nM;克隆適體F23靈敏度的結(jié)合測(cè)定,細(xì)菌數(shù)可低至2×106;通過ELADA和DFA法檢測(cè)F23與非發(fā)酵菌特異性結(jié)合,發(fā)現(xiàn)依次為銅綠假單胞菌、鮑曼不動(dòng)桿菌和嗜麥芽窄食單胞菌,這兩個(gè)方法的檢測(cè)結(jié)果是一致。 5.通過熒光基團(tuán)5’-FAM和5’-TAMRA標(biāo)記克隆適體F23對(duì)非發(fā)酵菌進(jìn)行熒光直接檢測(cè),結(jié)果發(fā)現(xiàn)F23與銅綠假單胞菌結(jié)合的熒光強(qiáng)度明顯比其它菌強(qiáng),尤其5’-TAMRA標(biāo)記適體更能夠減少組織自發(fā)性熒光的非特異性背景的干擾。 【結(jié)論】 本研究運(yùn)用指數(shù)富集配基的系統(tǒng)進(jìn)化(SELEX)技術(shù),利用其他非發(fā)酵菌進(jìn)行反篩,經(jīng)十五輪篩選出針對(duì)滅活銅綠假單胞菌的ssDNA適體,在國(guó)內(nèi)外首次運(yùn)用ELADA和DFA對(duì)篩選出的適體進(jìn)行親和力、靈敏度和特異性的分析,已成功篩選到高親和力高特異性的針對(duì)滅活銅綠假單胞菌的適體,并在國(guó)內(nèi)首次建立熒光基團(tuán)標(biāo)記適體快速鑒定銅綠假單胞菌檢測(cè)方法,為銅綠假單胞菌臨床快速鑒定帶來了希望。
[Abstract]:[Objective]
Using the phylogenetic (SELEX) technique of exponential enrichment ligand, the inactivated Pseudomonas aeruginosa was used as a compound target to screen out the high affinity and high specificity of Pseudomonas aeruginosa from 96 NT random oligonucleotide libraries synthesized in vitro, and the detection of Pseudomonas aeruginosa by a fluorescent group marker was established. Law.
[method]
1. a random sequence at both ends, a random sequence of 60 NT in the middle, a total length of 96 NT oligonucleotide libraries, and an inactivated Pseudomonas aeruginosa as the target, were screened by SELEX to screen out the high affinity and high specificity of Pseudomonas aeruginosa, and the Enzyme-linked aptamer direct assay, EL (EL) method was used to detect the high affinity and specificity of Pseudomonas aeruginosa. ADA) using digoxin anti digoxin antibody alkaline phosphatase system to carry out binding assay for ssDNA enrichment library and Pseudomonas aeruginosa.
2. in the experiment, we conducted the ssDNA Library in the twelfth and fourteenth rounds at the same time, respectively, to carry out two ideas of the ssDNA library and the screening and non screening of Acinetobacter maltophilia and Acinetobacter aeruginosa, and the indirect comparison of the binding force of the ssDNA enrichment library with Pseudomonas aeruginosa by ELADA.
3. The products from the 6th, 10th and 15th rounds of screening were amplified, purified, cloned and sequenced, and their primary and secondary structures were analyzed by related software.
4. the combination of cloned aptamers and Pseudomonas aeruginosa was determined by ELADA, and 3 cloned aptamers with high OD value were selected and their affinity was determined. Then, the sensitivity and specificity were further analyzed for the clones with the highest affinity. DFA (dot filtration assay, DFA) was used to make specific and non fermenting strains of F23 clones. Heterosexual testing.
5. a method for rapid identification of Pseudomonas aeruginosa by fluorescent group labeling was established: F23 suitable for 5 '-FAM and 5' -TAMRA fluorescent groups were labeled directly with non fermenting bacteria, washing, sealing, and fluorescence microscopy.
[results]
1. with the increase of the number of screening wheels, the OD value of Pseudomonas aeruginosa combined with the ssDNA enrichment library increased gradually. The color o value of the enrichment library after the combination of twelfth rounds of anti screening and Pseudomonas aeruginosa (0.448) was 23 times that of the first round of OD (0.022). When the binding site of the ssDNA enrichment library and Pseudomonas aeruginosa reached a saturation state, SsDNA adsorbed on Pseudomonas aeruginosa no longer increased.
2. there were significant differences in the OD value of the ssDNA enrichment library combined with Pseudomonas aeruginosa, which was screened and unscreened. The ratio of the lowest fifteenth round screen and the unscreened resieve was 1.3 times, to the highest fourteenth wheel ratio of 53 times.
The results of the 3. clone sequence found that the sixth rounds, the tenth and the fifteenth unscreened sequences were not enriched, and the clones of the fifteenth round screen were sequenced, and 2 sequences were enriched in 24 expected random sequences, almost identical (F23 and F47), and the homology was 97%. ClustalX and Mega2 software were used to analyze the homology and score of the first order structure of the sequence. Class, divided into 10 families (Family), using Mfold sever software to simulate two level structure.
4. cloned aptamer F17, F23 and F47 combined with Pseudomonas aeruginosa increased significantly, and the affinity determination showed that the Kd of F23 was 14.55nM, F47 was 77.46nM, F17 was 493.3nM, and the number of bacteria could be as low as 2 x 106 by the combination of the F23 sensitivity of cloned aptamer. Pseudomonas, Acinetobacter Bauman and Stenotrophomonas maltophilia, the results of the two methods are consistent.
5. fluorescence group 5 '-FAM and 5' -TAMRA were used to detect non fermenting bacteria by direct fluorescence detection. The results showed that the fluorescence intensity of F23 and Pseudomonas aeruginosa was stronger than that of other bacteria. Especially, 5 '-TAMRA labeled aptamer could reduce the nonspecific background interference of tissue spontaneous fluorescence.
[Conclusion]
In this study, we used the phylogenetic phylogenetic (SELEX) technology of exponential enrichment ligand to screen out other non fermenting bacteria and screened out the ssDNA suitable for the inactivated Pseudomonas aeruginosa by fifteen rounds. The affinity, sensitivity and specificity of the selected suitable bodies were analyzed by ELADA and DFA at home and abroad for the first time. The high affinity has been successfully screened. High specificity for the aptamer of Pseudomonas aeruginosa for inactivating the Pseudomonas aeruginosa, and for the first time to establish a rapid identification method for the identification of Pseudomonas aeruginosa by a fluorescent group labeling suitable for the first time in China, which has brought hope for the rapid identification of Pseudomonas aeruginosa.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R378.991
本文編號(hào)：2171594
[Abstract]:[Objective]
Using the phylogenetic (SELEX) technique of exponential enrichment ligand, the inactivated Pseudomonas aeruginosa was used as a compound target to screen out the high affinity and high specificity of Pseudomonas aeruginosa from 96 NT random oligonucleotide libraries synthesized in vitro, and the detection of Pseudomonas aeruginosa by a fluorescent group marker was established. Law.
[method]
1. a random sequence at both ends, a random sequence of 60 NT in the middle, a total length of 96 NT oligonucleotide libraries, and an inactivated Pseudomonas aeruginosa as the target, were screened by SELEX to screen out the high affinity and high specificity of Pseudomonas aeruginosa, and the Enzyme-linked aptamer direct assay, EL (EL) method was used to detect the high affinity and specificity of Pseudomonas aeruginosa. ADA) using digoxin anti digoxin antibody alkaline phosphatase system to carry out binding assay for ssDNA enrichment library and Pseudomonas aeruginosa.
2. in the experiment, we conducted the ssDNA Library in the twelfth and fourteenth rounds at the same time, respectively, to carry out two ideas of the ssDNA library and the screening and non screening of Acinetobacter maltophilia and Acinetobacter aeruginosa, and the indirect comparison of the binding force of the ssDNA enrichment library with Pseudomonas aeruginosa by ELADA.
3. The products from the 6th, 10th and 15th rounds of screening were amplified, purified, cloned and sequenced, and their primary and secondary structures were analyzed by related software.
4. the combination of cloned aptamers and Pseudomonas aeruginosa was determined by ELADA, and 3 cloned aptamers with high OD value were selected and their affinity was determined. Then, the sensitivity and specificity were further analyzed for the clones with the highest affinity. DFA (dot filtration assay, DFA) was used to make specific and non fermenting strains of F23 clones. Heterosexual testing.
5. a method for rapid identification of Pseudomonas aeruginosa by fluorescent group labeling was established: F23 suitable for 5 '-FAM and 5' -TAMRA fluorescent groups were labeled directly with non fermenting bacteria, washing, sealing, and fluorescence microscopy.
[results]
1. with the increase of the number of screening wheels, the OD value of Pseudomonas aeruginosa combined with the ssDNA enrichment library increased gradually. The color o value of the enrichment library after the combination of twelfth rounds of anti screening and Pseudomonas aeruginosa (0.448) was 23 times that of the first round of OD (0.022). When the binding site of the ssDNA enrichment library and Pseudomonas aeruginosa reached a saturation state, SsDNA adsorbed on Pseudomonas aeruginosa no longer increased.
2. there were significant differences in the OD value of the ssDNA enrichment library combined with Pseudomonas aeruginosa, which was screened and unscreened. The ratio of the lowest fifteenth round screen and the unscreened resieve was 1.3 times, to the highest fourteenth wheel ratio of 53 times.
The results of the 3. clone sequence found that the sixth rounds, the tenth and the fifteenth unscreened sequences were not enriched, and the clones of the fifteenth round screen were sequenced, and 2 sequences were enriched in 24 expected random sequences, almost identical (F23 and F47), and the homology was 97%. ClustalX and Mega2 software were used to analyze the homology and score of the first order structure of the sequence. Class, divided into 10 families (Family), using Mfold sever software to simulate two level structure.
4. cloned aptamer F17, F23 and F47 combined with Pseudomonas aeruginosa increased significantly, and the affinity determination showed that the Kd of F23 was 14.55nM, F47 was 77.46nM, F17 was 493.3nM, and the number of bacteria could be as low as 2 x 106 by the combination of the F23 sensitivity of cloned aptamer. Pseudomonas, Acinetobacter Bauman and Stenotrophomonas maltophilia, the results of the two methods are consistent.
5. fluorescence group 5 '-FAM and 5' -TAMRA were used to detect non fermenting bacteria by direct fluorescence detection. The results showed that the fluorescence intensity of F23 and Pseudomonas aeruginosa was stronger than that of other bacteria. Especially, 5 '-TAMRA labeled aptamer could reduce the nonspecific background interference of tissue spontaneous fluorescence.
[Conclusion]
In this study, we used the phylogenetic phylogenetic (SELEX) technology of exponential enrichment ligand to screen out other non fermenting bacteria and screened out the ssDNA suitable for the inactivated Pseudomonas aeruginosa by fifteen rounds. The affinity, sensitivity and specificity of the selected suitable bodies were analyzed by ELADA and DFA at home and abroad for the first time. The high affinity has been successfully screened. High specificity for the aptamer of Pseudomonas aeruginosa for inactivating the Pseudomonas aeruginosa, and for the first time to establish a rapid identification method for the identification of Pseudomonas aeruginosa by a fluorescent group labeling suitable for the first time in China, which has brought hope for the rapid identification of Pseudomonas aeruginosa.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R378.991
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 王雷;嗜水氣單胞菌和遲緩愛德華菌適體的SELEX篩選[D];集美大學(xué);2012年
，本文編號(hào)：2171594
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