体外SELEX技术筛选SpA适配子的研究及初步应用

发布时间：2018-01-19 14:25

  本文关键词： 指数富集配基的系统进化技术 SpA 适配子 荧光标记适配子直接检测法 磁珠　出处：《第二军医大学》2012年博士论文　论文类型：学位论文

【摘要】：【目的】 SpA是金黄色葡萄球菌的重要毒力因子，它在金葡菌抗吞噬细胞吞噬、诱导分泌天然抗体的B细胞凋亡、引起葡萄菌性肺炎、心内膜炎等方面发挥重要作用。本研究旨在利用指数富集配基的系统进化(SELEX)技术，以磁珠结合的SpA作为靶分子，从体外合成的96个nt的随机寡核苷酸文库中筛选出与SpA高亲和力、高特异性结合的适配子；并建立生物素标记适配子SpA的ELISA的检测方法；寻找抑制SpA功能位点适配子，达到控制金黄色葡萄球菌毒力的效果。 【方法】 1.将SpA包被于Dynal磁珠，并用BCA法检测SpA的包被效率，用免疫吸附法检测SpA包被磁珠后结构是否改变。 2.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，以磁珠包被的SpA为靶标，以磁力架分离结合与未结合的ssDNA，通过不断增加筛选的强度（增加tRNA量、缩短结合时间、增加震荡频率等），经SELEX筛选，筛选与SpA高亲和力、高特异性结合的适配子。 3.反筛：在第7轮、第9轮和第12轮筛选前，用ssDNA文库与空白磁珠结合，以未与空白磁珠结合的ssDNA文库做为筛选库，与SpA包被磁珠结合，以除去与SpA非特异性结合的ssDNA。 4.用荧光集团标记ssDNA作为筛选文库，应用TBS－380荧光定量仪测定2、4、6、7、9、10、11、12轮总投入库与未与SpA结合的ssDNA的荧光值，计算出各轮总投入库和筛选后与SpA结合的ssDNA量，观察ssDNA的富集情况。 5.将最后一轮筛选产物分别进行扩增、纯化、TA克隆、“蓝-白”斑选择、测序，得到适配子的核酸序列，并用相关软件分析适配子的一级和二级结构，了解适配子的各结构特点并根据序列相似性和自由能挑选适配子家族。 6.用Dot-blot方法测定适配子家族与SpA的结合情况。 7.生物素标记适配子，根据SpA-适配子-生物素-亲合素-碱性磷酸酶-底物结合反应步骤，用适配子ELISA方法测定不同浓度适配子与SpA的结合情况，并用GraphPad Prism软件通过非线性回归分析获得适配子的解离常数。 8.将适配子标记胶体金颗粒，将靶物质点样于硝酸纤维素膜，用斑点免疫金渗滤法分析适配子与SpA结合的特异性。 9.建立适配子ELISA检测SpA方法，并建立标准曲线。 10.建立SpA结合的磁珠IgG吸附方法，观察适配子对SpA与IgG结合的抑制。 11.从细胞学角度，根据细菌或SpA刺激细胞产生的IL-8和P38磷酸化特性，观察适配子对金黄色葡萄球菌和SpA的抑制情况。 12.用小鼠做体内实验，观察适配子作用下，金黄色葡萄球菌感染小鼠肺炎、菌血症、死亡等事件发生情况，观察金黄色葡萄球菌感染或SpA作用小鼠，肺内白细胞的募集情况。 【结果】 1. SpA高效率包被磁珠，包被效率为88.18%；并且包被后不影响SpA与IgG结合。 2.随着筛选轮数的增加, ssDNA富集库与SpA结合的荧光值逐渐增加,第十二轮筛选获得的富集库与SpA结合率45.6%是第一轮2.1%的21.7倍。到第九轮，ssDNA富集库与SpA的结合达到基本饱和状态时,吸附到SpA上的ssDNA不再增加。 3.48个克隆序列结果显示，共有45个菌落得到结果，根据软件分析，所有序列二级结构都以茎环、发夹结构为主，根据序列和结构，共挑选出五个家族做进一步分析。各家族间同源性大都在70%以下。 4.经Dot-blot方法检测发现，第一、第二、第三适配子家族与SpA结合的阳性结果最强。进行亲和力测定，四个家族的Kd值分别为：17.31±6.24nM；25.22±7.51nM、24.27±6.98nM、53.18±7.58nM；通过胶体金渗滤法检测发现第一、第二、第三家族均与SpA特异性结合。 5.通过用生物素标记适配子，建立了第二家族适配子ELISA方法，并建立了SpA做定量检测标准曲线。 6.通过免疫吸附抑制实验发现，第一家庭适配子可以明显抑制IgG与SpA的结合，使SpA结合IgG的比例从35.0%降至10.7%。 7.金黄色葡萄球菌或SpA与表皮细胞TNFR作用促进P38磷酸化，并最终导致细胞IL-8分泌增加，加入第一家族适配子后，细菌作用下细胞IL-8浓度从971.67±359.40pg/ml减少到487.08±189.09pg/ml（P=0.0013），SpA作用下细胞IL-8浓度从778.08±278.52pg/ml减少到255.58±136.76pg/ml（P0.0001）。表皮细胞P38磷酸化增加。 8.小鼠金黄色葡萄球菌感染模型中，同时加入适配子与细菌或适配子与SpA使小鼠肺炎、菌血症发生率明显下降，分别从58.33下降到16.67（肺炎）(p=0.0429)和25%（菌血症）(p=0.0316)，肺内白细胞募集情况也得到改善，分别从58.08%±30.55%下降到29.83%±16.44%（细菌）（P0.0001）；从34%±9%下降到16%±5%（SpA）(p=0.0028)。 【结论】 本研究运用指数富集配基的系统进化(SELEX)技术，用磁珠做为分离体系，用排除非特异结合的反筛，经十二轮筛选，在国内、外最先筛选出针对SpA的ssDNA适配子，并首次运用DOT-BLOT方法测定适配子与SpA的结合，建立SpA的适配子检测方法。本研究还找到了SpA功能抑制适配子，，初步证明了适配子对SpA功能位点的封闭作用，并探讨了适配子通过对SpA的功能抑制而在对抗细菌感染中的作用，为今后寻找新的抗金黄色葡萄球菌药物靶点奠定了基础。
[Abstract]:[Objective]
SpA is an important virulence factor of Staphylococcus aureus and its anti phagocytosis in Staphylococcus aureus, B cell apoptosis induced by natural antibody secretion caused by Staphylococcus, pneumonia, endocarditis and other aspects play an important role. The purpose of this study is to use the systematic evolution of ligands by exponential enrichment (SELEX) technology, with the magnetic beads combined with SpA as the target molecule from random oligonucleotide library 96 NT synthesis in vitro screened in SpA with high affinity aptamers specific binding; and establish a method for determination of biotin labeled aptamers SpA ELISA; for inhibition of SpA functional sites of aptamer, to control Staphylococcus aureus virulence effect.
[method]
1. the SpA package was given to the Dynal magnetic bead and the BCA method was used to detect the efficiency of the SpA package. The structure of the SpA wrapped magnetic beads was detected by the immunoadsorption method.
2. in vitro construction at both ends fixed sequence, intermediate random sequence of 60 NT, 96 NT full-length oligonucleotide library, using magnetic beads coated with ssDNA SpA as the target, and separation of unbound by magnetic frame, by increasing the intensity of screening (increased tRNA amount, shorten the bonding time, increase the shock the frequency, etc.) by SELEX screening, screening and SpA with high affinity, high specific binding of the aptamers.
3., reverse sieving: before the seventh round, Ninth rounds and twelfth rounds of screening, the ssDNA library was combined with the blank magnetic beads, the ssDNA library which was not combined with the blank magnetic beads was selected as the screening library, and it was combined with the SpA coated magnetic beads to remove the ssDNA. which was not specifically combined with SpA.
4., fluorescent group labeling ssDNA was used as the screening library. The fluorescence value of ssDNA which was combined with 2,4,6,7,9,10,11,12 SpA and SpA did not be measured by TBS 380 fluorescence quantitative analyzer. The ssDNA content of the total input database and SpA after screening was calculated, and the enrichment of ssDNA was observed.
5. will be the last round of screening products were amplified, purified, cloned TA, "blue and white" spot selection, sequencing, get the nucleic acid sequence of aptamer, and analysis of aptamers one grade and two grade structure by using related software, to understand the structural characteristics of aptamers and according to sequence similarity and freedom to choose aptamer family.
6. the Dot-blot method was used to determine the combination of the aptamer family and the SpA.
7. biotin labeled aptamers, according to the SpA- aptamer - biotin avidin alkaline phosphatase substrate binding reaction steps were determined by combination of different concentrations of aptamers and SpA aptamer ELISA method and nonlinear regression analysis to obtain the dissociation constant of aptamers by using GraphPad Prism software.
8. the colloid gold particles were labeled by the aptamer, the target substance was sampled from the nitrocellulose membrane, and the speckle immunogold filtration assay was used to analyze the specificity of the binding of the aptamer to the SpA.
9. an aptamer ELISA was established to detect the SpA method and the standard curve was established.
10. a SpA binding magnetic bead IgG adsorption method was established to observe the inhibition of the binding of SpA to IgG by the aptamer.
11. from the cytological point of view, the inhibition of Staphylococcus aureus and SpA by the aptamer was observed on the basis of the phosphorylation of IL-8 and P38 produced by bacteria or SpA stimulated cells.
12., in vivo experiment with mice, we observed the incidence of pneumonia, bacteremia and death of Staphylococcus aureus infected with aptamers under the action of aptamers, and observed the recruitment of white blood cells in mice infected with Staphylococcus aureus or SpA.
[results]
The 1. SpA high efficiency package was coated with magnetic beads, and the envelope was efficiently 88.18%; and the package was not affected by the combination of SpA and IgG.
2. with the increase of the number of rounds of screening, fluorescent ssDNA enriched library combined with SpA value increased gradually, the twelfth round of screening the enrichment library and the SpA binding rate of 45.6% is 21.7 times the first round of 2.1%. To the ninth round, combined with ssDNA enrichment library and SpA reached saturation, adsorption to SpA ssDNA no longer increased.
The results of 3.48 cloning sequences showed that 45 colonies were obtained. According to the software analysis, two rank structures of all sequences were mainly stem ring and hairpin structure. According to the sequence and structure, five families were selected for further analysis. The homology among the families was mostly below 70%.
After 4. Dot-blot, the first, second, third. The positive results of gamete family SpA combined with the strongest affinity. Determination of four family Kd = 17.31 + 6.24nM; 25.22 + 7.51nM, 24.27 + 6.98nM, 53.18 + 7.58nM; by colloidal gold filtration assay found first, second. The third families were combined with specific SpA.
5. by using biotin to mark the aptamer, a second family aptamer ELISA method was established, and a standard curve for quantitative detection of SpA was established.
6. through the immunosorbent inhibition experiment, it was found that the first family aptamer could obviously inhibit the combination of IgG and SpA, and reduced the proportion of SpA to IgG from 35% to 10.7%.
7. Staphylococcus aureus or SpA and epidermal cells TNFR promotes the phosphorylation of P38, and eventually lead to increased secretion of IL-8 cells, with the first family of aptamers, bacterial cells IL-8 + concentration from 971.67 359.40pg/ml reduced to 487.08 + 189.09pg/ml (P=0.0013), SpA cells IL-8 + 278.52pg/ml concentrations from 778.08 reduced to 255.58 (P0.0001 + 136.76pg/ml). Increased P38 phosphorylation of epidermal cells.
8. mice infected with Staphylococcus aureus in the model, while adding aptamers with bacteria or aptamers and SpA mice Fei Yan, the incidence of bacteremia was significantly decreased, respectively, decreased from 58.33 to 16.67 (Fei Yan) (p=0.0429) and 25% (bacteremia) (p=0.0316), pulmonary leukocyte recruitment has also been improved, respectively, down to 29.83% + 16.44% + 30.55% (bacteria) from 58.08% (P0.0001); reduced from 34% + 9% to 16% + 5% (SpA) (p=0.0028).
[Conclusion]
On the basis of systematic evolution of ligands by exponential enrichment (SELEX) technology, using magnetic beads as separate systems, with exclusion of anti screen non-specific binding, after twelve rounds of screening, in the domestic, abroad first screened for SpA ssDNA aptamer, and the first use of the DOT-BLOT method for the determination of SpA and combining with the adaptation. The establishment of aptamer detection methods of SpA. This study also found the inhibition of SpA aptamer, proved the blocking effect of aptamers for SpA functional sites, and discusses the aptamer inhibition against bacterial infection through the SpA function, lays the foundation for finding new anti Staphylococcus aureus drug targets.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378

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