本文關(guān)鍵詞： 指數(shù)富集配基的系統(tǒng)進化技術(shù) SpA 適配子 熒光標記適配子直接檢測法 磁珠　出處：《第二軍醫(yī)大學》2012年博士論文　論文類型：學位論文

【摘要】：【目的】 SpA是金黃色葡萄球菌的重要毒力因子，它在金葡菌抗吞噬細胞吞噬、誘導分泌天然抗體的B細胞凋亡、引起葡萄菌性肺炎、心內(nèi)膜炎等方面發(fā)揮重要作用。本研究旨在利用指數(shù)富集配基的系統(tǒng)進化(SELEX)技術(shù)，以磁珠結(jié)合的SpA作為靶分子，從體外合成的96個nt的隨機寡核苷酸文庫中篩選出與SpA高親和力、高特異性結(jié)合的適配子；并建立生物素標記適配子SpA的ELISA的檢測方法；尋找抑制SpA功能位點適配子，達到控制金黃色葡萄球菌毒力的效果。 【方法】 1.將SpA包被于Dynal磁珠，并用BCA法檢測SpA的包被效率，用免疫吸附法檢測SpA包被磁珠后結(jié)構(gòu)是否改變。 2.在體外構(gòu)建兩端為固定序列、中間為60個nt的隨機序列、全長96個nt的寡核苷酸文庫，以磁珠包被的SpA為靶標，以磁力架分離結(jié)合與未結(jié)合的ssDNA，通過不斷增加篩選的強度（增加tRNA量、縮短結(jié)合時間、增加震蕩頻率等），經(jīng)SELEX篩選，篩選與SpA高親和力、高特異性結(jié)合的適配子。 3.反篩：在第7輪、第9輪和第12輪篩選前，用ssDNA文庫與空白磁珠結(jié)合，以未與空白磁珠結(jié)合的ssDNA文庫做為篩選庫，與SpA包被磁珠結(jié)合，以除去與SpA非特異性結(jié)合的ssDNA。 4.用熒光集團標記ssDNA作為篩選文庫，應(yīng)用TBS－380熒光定量儀測定2、4、6、7、9、10、11、12輪總投入庫與未與SpA結(jié)合的ssDNA的熒光值，計算出各輪總投入庫和篩選后與SpA結(jié)合的ssDNA量，觀察ssDNA的富集情況。 5.將最后一輪篩選產(chǎn)物分別進行擴增、純化、TA克隆、“藍-白”斑選擇、測序，得到適配子的核酸序列，并用相關(guān)軟件分析適配子的一級和二級結(jié)構(gòu)，了解適配子的各結(jié)構(gòu)特點并根據(jù)序列相似性和自由能挑選適配子家族。 6.用Dot-blot方法測定適配子家族與SpA的結(jié)合情況。 7.生物素標記適配子，根據(jù)SpA-適配子-生物素-親合素-堿性磷酸酶-底物結(jié)合反應(yīng)步驟，用適配子ELISA方法測定不同濃度適配子與SpA的結(jié)合情況，并用GraphPad Prism軟件通過非線性回歸分析獲得適配子的解離常數(shù)。 8.將適配子標記膠體金顆粒，將靶物質(zhì)點樣于硝酸纖維素膜，用斑點免疫金滲濾法分析適配子與SpA結(jié)合的特異性。 9.建立適配子ELISA檢測SpA方法，并建立標準曲線。 10.建立SpA結(jié)合的磁珠IgG吸附方法，觀察適配子對SpA與IgG結(jié)合的抑制。 11.從細胞學角度，根據(jù)細菌或SpA刺激細胞產(chǎn)生的IL-8和P38磷酸化特性，觀察適配子對金黃色葡萄球菌和SpA的抑制情況。 12.用小鼠做體內(nèi)實驗，觀察適配子作用下，金黃色葡萄球菌感染小鼠肺炎、菌血癥、死亡等事件發(fā)生情況，觀察金黃色葡萄球菌感染或SpA作用小鼠，肺內(nèi)白細胞的募集情況。 【結(jié)果】 1. SpA高效率包被磁珠，包被效率為88.18%；并且包被后不影響SpA與IgG結(jié)合。 2.隨著篩選輪數(shù)的增加, ssDNA富集庫與SpA結(jié)合的熒光值逐漸增加,第十二輪篩選獲得的富集庫與SpA結(jié)合率45.6%是第一輪2.1%的21.7倍。到第九輪，ssDNA富集庫與SpA的結(jié)合達到基本飽和狀態(tài)時,吸附到SpA上的ssDNA不再增加。 3.48個克隆序列結(jié)果顯示，共有45個菌落得到結(jié)果，根據(jù)軟件分析，所有序列二級結(jié)構(gòu)都以莖環(huán)、發(fā)夾結(jié)構(gòu)為主，根據(jù)序列和結(jié)構(gòu)，共挑選出五個家族做進一步分析。各家族間同源性大都在70%以下。 4.經(jīng)Dot-blot方法檢測發(fā)現(xiàn)，第一、第二、第三適配子家族與SpA結(jié)合的陽性結(jié)果最強。進行親和力測定，四個家族的Kd值分別為：17.31±6.24nM；25.22±7.51nM、24.27±6.98nM、53.18±7.58nM；通過膠體金滲濾法檢測發(fā)現(xiàn)第一、第二、第三家族均與SpA特異性結(jié)合。 5.通過用生物素標記適配子，建立了第二家族適配子ELISA方法，并建立了SpA做定量檢測標準曲線。 6.通過免疫吸附抑制實驗發(fā)現(xiàn)，第一家庭適配子可以明顯抑制IgG與SpA的結(jié)合，使SpA結(jié)合IgG的比例從35.0%降至10.7%。 7.金黃色葡萄球菌或SpA與表皮細胞TNFR作用促進P38磷酸化，并最終導致細胞IL-8分泌增加，加入第一家族適配子后，細菌作用下細胞IL-8濃度從971.67±359.40pg/ml減少到487.08±189.09pg/ml（P=0.0013），SpA作用下細胞IL-8濃度從778.08±278.52pg/ml減少到255.58±136.76pg/ml（P0.0001）。表皮細胞P38磷酸化增加。 8.小鼠金黃色葡萄球菌感染模型中，同時加入適配子與細菌或適配子與SpA使小鼠肺炎、菌血癥發(fā)生率明顯下降，分別從58.33下降到16.67（肺炎）(p=0.0429)和25%（菌血癥）(p=0.0316)，肺內(nèi)白細胞募集情況也得到改善，分別從58.08%±30.55%下降到29.83%±16.44%（細菌）（P0.0001）；從34%±9%下降到16%±5%（SpA）(p=0.0028)。 【結(jié)論】 本研究運用指數(shù)富集配基的系統(tǒng)進化(SELEX)技術(shù)，用磁珠做為分離體系，用排除非特異結(jié)合的反篩，經(jīng)十二輪篩選，在國內(nèi)、外最先篩選出針對SpA的ssDNA適配子，并首次運用DOT-BLOT方法測定適配子與SpA的結(jié)合，建立SpA的適配子檢測方法。本研究還找到了SpA功能抑制適配子，，初步證明了適配子對SpA功能位點的封閉作用，并探討了適配子通過對SpA的功能抑制而在對抗細菌感染中的作用，為今后尋找新的抗金黃色葡萄球菌藥物靶點奠定了基礎(chǔ)。
[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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R378

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