体外SELEX技术筛选灭活铜绿假单胞菌适体的研究及初步应用
发布时间：2018-08-08 11:05
【摘要】： 【目的】 利用指数富集配基的系统进化(SELEX)技术,以灭活的铜绿假单胞菌作为复合靶分子,从体外合成的96个nt的随机寡核苷酸文库中筛选出与铜绿假单胞菌高亲和力高特异性的适体,并建立荧光基团标记适体快速鉴定铜绿假单胞菌的检测方法。 【方法】 1.在体外构建两端为固定序列,中间为60个nt的随机序列,全长96个nt的寡核苷酸文库,以灭活的铜绿假单胞菌为靶标,经SELEX筛选,筛选出针对铜绿假单胞菌高亲和力高特异性的适体,用酶连接适体直接检测法(Enzyme-linked aptamer direct assay,ELADA)利用地高辛-抗地高辛抗体-碱性磷酸酶系统进行ssDNA富集库与铜绿假单胞菌的结合测定。 2.我们在实验中从第12轮和第14轮分别同时进行ssDNA文库与嗜麦芽窄食单胞菌和鲍曼不动杆菌反筛和未反筛的两条思路,运用ELADA间接比较反筛和未反筛获得的ssDNA富集库与铜绿假单胞菌的结合力。 3.将第6轮、第10轮和第15轮筛选产物分别进行扩增、纯化, TA克隆、测序并用相关软件分析其一级和二级结构。 4.用ELADA进行克隆适体与铜绿假单胞菌的结合测定,选出OD值明显高的3个克隆适体,并对其进行亲和力测定;然后对亲和力最高的克隆适体,进一步进行灵敏度和特异性分析。运用DFA(dot filtration assay, DFA)对F23克隆适体与非发酵菌进行特异性检测。 5.建立荧光基团标记适体快速鉴定铜绿假单胞菌检测方法:将F23适体进行5’-FAM和5’-TAMRA荧光基团标记直接和非发酵菌结合、洗涤、封片,荧光显微镜检查。 【结果】 1.随着筛选轮数的增加,铜绿假单胞菌与ssDNA富集库结合的OD值逐渐增加,第十二轮反筛选获得的富集库与铜绿假单胞菌结合后显色的OD值(0.448)是第一轮OD值(0.022)的23倍。当ssDNA富集库与铜绿假单胞菌上的结合位点达到一个饱和状态时,吸附到铜绿假单胞菌上的ssDNA就不再增加。 2.经过反筛和未反筛的ssDNA富集库与铜绿假单胞菌结合的OD值有显著差异,从最低的第15轮的反筛和未反筛的OD比值为1.3倍,到最高的第14轮比值的53倍。 3.克隆序列结果发现第6轮、第10轮和第15轮未反筛的序列均无富集,而第15轮反筛的克隆子进行测序,发现24个预期随机序列中有2条序列富集,几乎完全一致(F23和F47),同源性达97%,利用ClustalX和Mega2软件分析序列的一级结构的同源性和分类,分成10个家族(Family),利用Mfold sever软件模拟二级结构。 4.克隆适体F17、F23和F47与铜绿假单胞菌结合的OD值明显增高;进行亲和力测定,可知:F23的Kd为14.55nM,F47为77.46nM,F17为493.3nM;克隆适体F23灵敏度的结合测定,细菌数可低至2×106;通过ELADA和DFA法检测F23与非发酵菌特异性结合,发现依次为铜绿假单胞菌、鲍曼不动杆菌和嗜麦芽窄食单胞菌,这两个方法的检测结果是一致。 5.通过荧光基团5’-FAM和5’-TAMRA标记克隆适体F23对非发酵菌进行荧光直接检测,结果发现F23与铜绿假单胞菌结合的荧光强度明显比其它菌强,尤其5’-TAMRA标记适体更能够减少组织自发性荧光的非特异性背景的干扰。 【结论】 本研究运用指数富集配基的系统进化(SELEX)技术,利用其他非发酵菌进行反筛,经十五轮筛选出针对灭活铜绿假单胞菌的ssDNA适体,在国内外首次运用ELADA和DFA对筛选出的适体进行亲和力、灵敏度和特异性的分析,已成功筛选到高亲和力高特异性的针对灭活铜绿假单胞菌的适体,并在国内首次建立荧光基团标记适体快速鉴定铜绿假单胞菌检测方法,为铜绿假单胞菌临床快速鉴定带来了希望。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.991
本文编号：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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.991
【引证文献】
相关硕士学位论文 前1条
1 王雷;嗜水气单胞菌和迟缓爱德华菌适体的SELEX筛选[D];集美大学;2012年
，本文编号：2171594
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