發(fā)布時間：2018-06-13 17:45

  本文選題：誘導性 + 報告基因載體��； 參考：《復旦大學》2011年博士論文

【摘要】：1944年以來,氨基糖苷類抗生素臨床應用走過了漫長的道路,細菌也產(chǎn)生了更強的耐藥性。氨基糖苷類抗生素耐藥機制包括：抗生素乙�；�,腺苷化或磷酸化；細菌外膜通透性改變,使胞內(nèi)抗生素濃度降低,減少內(nèi)膜運輸,主動外排,藥物誘捕；30S核糖體亞基作用位點突變；抗生素結合位點甲基化等。而誘導抗性基因的表達成為當前抗生素耐藥的主要問題。 AAC(6')-IIb位于熒光假單胞菌的染色體上,該基因表達使細菌對慶大霉素、妥布霉素、奈替米星、地貝卡星和西索米星產(chǎn)生抗性。我們命名其5'-UTR非翻譯區(qū)序列為aac, aac可翻譯成短肽,具有ermC、fexA一樣的性質(zhì)。vienna RNA結構在線分析顯示,aac在不同溫度下呈現(xiàn)不同結構,說明其不同結構可能會影響AAC(6’)-IIb的表達。為研究aac的功能,構建報告基因pGEX-aac-lacZa表達載體,選用具有代表性的氨基糖苷類結構模型kanamycin B, lacZ酶活顯示加入kanamycin B組酶活升高,證實其能誘導lacZa的表達。而熒光定量PCR發(fā)現(xiàn)lacZa轉錄水平上,加抗生素組比對照組有稍微上調(diào),推測可能是mycin B與aac轉錄產(chǎn)物作用后,穩(wěn)定aac的結構進而導致lacZa的稍微上調(diào)。不同濃度kanamycin B不引起lacZa轉錄產(chǎn)物的變化,推測kanamycin B組與對照組aac-lacZa實際上沒有轉錄產(chǎn)物表達變化,只有aac轉錄產(chǎn)物二級結構的變化。neomycin與gentamycin進一步驗證lacZ酶活與lacZa轉錄產(chǎn)物,結果與kanamycinB相同,推測氨基糖苷類抗生素能與aac結合,進而誘導lacZa的表達。 平板誘導實驗直觀展示lacZa表達情況,除spectinomycin外所用13種氨基糖苷類抗生素均能誘導報告基因表達,推測spectinomycin結構中沒有氨基或者其結構未與aac結合,進而導致lacZa表達無變化。SPR實驗驗證aac RNA結合,并獲得結合常數(shù)。SPR實驗需要aac RNA3’末端標記生物素,經(jīng)驗證標記過程RNA無明顯降解,標記效率高,產(chǎn)量多。進一步體外SPR實驗證明,aac RNA能與除spectinomycin外氨基糖苷類抗生素結合,而結合常數(shù)與抗生素結構中氨基位置和結構有關。RNA與小分子結合調(diào)控相關基因表達為“核開關”的定義,而ermC、fexA翻譯后停靠調(diào)控具體機制仍未知,因為在體外前導肽不能與相對應抗生素結合,因此核開關理論可能適合ermC、fexA(?)(?)aac。 構建pGEX-RmtB-aac-lacZa報告載體,16S rRNA甲基化,平板誘導實驗顯示除spectinomycin外氨基糖苷類抗生素仍能誘導lacZa的翻譯,證明aac與氨基糖苷類抗生素直接結合,aac是以氨基糖苷類抗生素為配體的核開關。抗性基因表達菌株誘導環(huán)狀大小不明顯,推測修飾后的核糖體影響了lacZa的翻譯,而所需抗生素誘導濃度較高,推測16SrRNA甲基化后氨基糖苷類抗生素結合aac的體內(nèi)濃度降低,具體機制需進一步研究。 aac作為核開關,其與氨基糖苷類抗生素結合前后應有結構改變。SHAPE實驗證實,氨基糖苷類抗生素與aac RNA結合后使翻譯起始SD-2及AUG-2結構打開,而spectinomycin不與之結合。通過vienna在線預測RNA結構及CLUSTALW同源性比對分析發(fā)現(xiàn),aac結構中存在一段保守序列AGUC與16SrRNA相同,推測結合位點可能發(fā)生在相關位置。經(jīng)SHAPE數(shù)據(jù)分析發(fā)現(xiàn),靠近AGUC位置的“CCC"序列信號下降,證明其可能與氨基糖苷類抗生素結合,因此推測其為氨基糖苷類抗生素的結合位點。通過數(shù)據(jù)分析,預測出aac加抗生素前后的結構,而參與結合位點的反應基團還有待進一步研究。 綜上所述,本課題發(fā)現(xiàn)一個新核開關aac,配體為氨基糖苷類抗生素,這也是首次報導抗生素為配體的核開關,為抗性基因誘導表達的機理研究開辟了一個新的領域。 硫化氫是第三大氣體信號分子,參與血管張力,心肌收縮,神經(jīng)傳遞和胰島素的分泌調(diào)節(jié)。當體內(nèi)缺少硫化氫時,觀察到動脈和肺動脈高壓,阿爾茨海默病,胃粘膜損傷和各種動物模型肝硬化。外源硫化氫可改善心肌功能障礙與缺血相關／再灌注損傷,降低胃粘膜損傷。另一方面,對其過度表達可能導致炎癥性疾病,感染性休克,腦中風,唐氏綜合征患者心理障礙的發(fā)病,其表達的減少可能對這些疾病的治療有潛在價值。 目前對硫化氫的研究還處于初步階段,對硫化氫作用的分子機制及直接作用的靶分子至今不明。為了闡明硫化氫細胞效應的分子機制與調(diào)控網(wǎng)絡,并進一步通過硫化氫途徑干預重大疾病尋找新的靶點,我們運用裂殖酵母生物芯片的方法,選用覆蓋全基因組編碼序列的裂殖酵母的生物芯片,對比在細胞內(nèi)有硫化氫和沒有硫化氫的條件下全基因組表達譜的差異,為分離出參與細胞周期,細胞增殖和凋亡過程中信號通路中的重要蛋白因子做鋪墊。 經(jīng)過相關實驗驗證和分析,采用50μM硫氫化鈉為硫化氫的供體,處理后的裂殖酵母進行全基因組RNA提取,反轉錄后基因芯片雜交。信號采用SAS分析系統(tǒng)標準化,2-fold, t-test分析篩選到83個差異表達基因,1.5-fo1d篩選到280個差異基因。經(jīng)cluster分析發(fā)現(xiàn),三個生物重復實驗具有重復性,實驗結論可靠。而采取的1.2-fold變化基因的實時熒光定量PCR分析發(fā)現(xiàn),基因芯片數(shù)據(jù)可信度高,所以我們采用1.5-fold為主要分析所用基因。 采用DAVID在線軟件對差異表達基因進行功能分類,默認設置(counts2, EASE score＜0.1),2-fold基因GO生物過程分類顯示,有20個下調(diào)基因,63個基因上調(diào)。1.5—fold的基因中,有156個基因表達上調(diào),124個基因表達下調(diào)。其中2-fold中31個基因與應激反應相關,18個基因編碼預測的或已知的運輸?shù)鞍?11個基因編碼與細胞周期/減數(shù)分裂相關的蛋白質(zhì),10個基因編碼的蛋白質(zhì)參與氧化還原反應。 與應激相關基因的比較發(fā)現(xiàn),硫化氫誘導基因中46%的基因與過氧化氫相同,43%的基因與鎘相同,49%的基因與熱相同。然而,只有12%和24%硫化氫誘導基因與山梨醇和甲基甲烷磺鹽相同。與過氧化氫相同基因可能說明硫化氫可以保護細胞免受氧化應激,參與MAPK信號通路中的一些基因的表達變化可能是硫化氫的靶基因。 在全基因組蛋白定位研究中,4954個基因中的480個基因定位在線粒體(9.6%基因定位在線粒體)。大于1.5倍基因中124個基因下調(diào),而其中定位在線粒體的基因有23個受硫化氫處理下調(diào)(18.5%基因定位于線粒體)。表明硫化氫引起許多線粒體基因表達下調(diào),可能對線粒體功能的有影響。 通過檢測裂殖酵母的呼吸中的耗氧量和線粒體膜電位來驗證硫化氫對線粒體功能的影響,實驗結果表明,硫化氫處理后的裂殖酵母耗氧量降低,線粒體膜電位下降,證明線粒體功能損傷,這充分印證基因芯片數(shù)據(jù)分析結果,硫化氫引起線粒體基因表達下調(diào)。 硫化氫引起的一些基因差異表達可為人類同源蛋白質(zhì)研究和分子靶點提供線索,為進一步探索與硫化氫相關的人類疾病機理和治療提供基礎。
[Abstract]:Since 1944, the clinical application of aminoglycoside antibiotics has come a long way, and bacteria have also produced stronger resistance. The mechanisms of aminoglycoside resistance include antibiotic acetylation, adenosine or phosphorylation, bacterial membrane permeability change, reduction of intracellular antibiotic concentration, reduction of endometrium transport, active efflux, drugs Trapping, 30S ribosome subunit mutation and methylation of antibiotic binding sites, and the expression of inducible resistance genes are the main problems of antibiotic resistance at present.
AAC (6') -IIb is located on the chromosomes of Pseudomonas fluorescens, which makes bacteria resistant to gentamicin, tobramycin, netilmicin, netilmicin, and sisomicin. We named its 5'-UTR untranslated region sequence as AAC, AAC can be translated into short peptide, ermC, fexA like properties of.Vienna RNA structure online analysis show that AAC is in AAC. Different structures show different structures, indicating that different structures may affect the expression of AAC (6 ') -IIb. In order to study the function of AAC, the pGEX-aac-lacZa expression vector of the reporter gene is constructed, the representative amino glycoside structure model kanamycin B is selected, and the activity of lacZ enzyme activity is added into the B group of kanamycin B, which proves that it can induce lacZa. The fluorescence quantitative PCR found that at lacZa transcriptional level, the antibiotic group was slightly up-regulated than the control group. It was presumed that after the action of mycin B and AAC transcripts, the structure of the AAC was stable and the lacZa was slightly up-regulated. The kanamycin B of different concentrations did not cause the change of the lacZa transcript, and the kanamycin B group and the control group were presumed to be true. There is no change in the expression of transcriptional products. Only the changes in the two grade structure of the AAC transcriptional products.Neomycin and gentamycin further verify the lacZ enzyme activity and the lacZa transcript, and the results are the same as kanamycinB. It is presumed that the aminoglycoside antibiotics can bind to AAC and then induce the expression of lacZa.
The flat plate induction test shows the expression of lacZa intuitively. 13 aminoglycoside antibiotics except spectinomycin can induce the expression of the reporter gene. It is speculated that there is no amino group in the structure of spectinomycin or its structure is not combined with AAC, which leads to the AAC RNA binding without change of.SPR in lacZa expression, and the.SPR experiment of binding constant is obtained. It is necessary to mark biotin at the end of AAC RNA3 ', which has no obvious degradation, and the labeling efficiency is high and the yield is high. Further in vitro SPR experiment has proved that AAC RNA can combine with spectinomycin amidoside antibiotics, and the binding constant is associated with the binding of the amino position and structure of the amino group and structure in the structure of the antibiotic and the binding of.RNA to small molecules in the structure of the amino group and the structure of the antibiotic. Because the expression is the definition of "nuclear switch", the specific mechanism of ermC, fexA after translation is still unknown, because the leading peptide in vitro can not be combined with relative antibiotics, so the theory of nuclear switch may be suitable for ermC, fexA (?) aac.
The pGEX-RmtB-aac-lacZa report carrier, 16S rRNA methylation, and the plate induction test showed that the spectinomycin external aminoglycoside antibiotics still could induce the translation of lacZa, which proved that AAC was directly combined with aminoglycoside antibiotics, and AAC was a nuclear switch with aminoglycoside antibiotics as the ligand. It is conjectured that the modified ribosome affects the translation of lacZa, and the concentration of antibiotics required is high. It is speculated that the concentration of aminoglycoside antibiotics combined with AAC after 16SrRNA methylation is reduced, and the specific mechanism needs to be further studied.
AAC as a nuclear switch, its structure changes.SHAPE experiment with aminoglycoside antibiotics before and after the combination of aminoglycoside antibiotics. The combination of aminoglycoside antibiotics and AAC RNA opens the translation initiation SD-2 and AUG-2 structure, while spectinomycin does not combine with them. The RNA structure and CLUSTALW homology comparison analysis of CLUSTALW are found to be found in AAC structure through Vienna online. The existence of a conservative sequence AGUC is the same as 16SrRNA, and the binding site may occur at the relevant position. The SHAPE data analysis shows that the "CCC" sequence signal near the AGUC position decreases, which proves that it may be combined with aminoglycoside antibiotics, thus speculates that it is the binding site of aminoglycoside antibiotics. By data analysis, the prediction of a The structure of AC plus and without antibiotics, and the reaction sites involved in binding sites need further study.
In summary, we have found a new nuclear switch AAC, a ligand of aminoglycoside antibiotics, which is the first to report the nuclear switch of the antibiotic as a ligand, which opens up a new field for the mechanism of resistant gene expression.
Hydrogen sulfide (H2S) is the third major gas signal molecule involved in vascular tension, myocardial contraction, neurotransmission and insulin secretion. When hydrogen sulfide is lacking in the body, arterial and pulmonary hypertension, Alzheimer's disease, gastric mucosal injury and various animal models of liver cirrhosis are observed. On the other hand, overexpression of it may lead to inflammatory diseases, septic shock, stroke, and mental disorders in Down's syndrome, and the decrease in expression may be of potential value for the treatment of these diseases.
At present, the study of hydrogen sulfide is still in the initial stage. The molecular mechanism of hydrogen sulfide and the target molecules that have direct action are unknown. In order to clarify the molecular mechanism and regulatory network of the hydrogen sulfide cell effect, and to further explore new targets by interfering with the major diseases through hydrogen sulfide pathway, we use the recipe of fission yeast biochip. Method, the biochip of fission yeast, which covers the whole genome encoding sequence, is used to compare the difference of the whole genome expression profiles under the conditions of hydrogen sulfide and hydrogen sulfide in the cells, and paving the important protein factors in the signal pathways involved in cell cycle, cell proliferation and apoptosis.
Through the experimental verification and analysis, 50 M sodium hydrogen sulfide was used as the donor of hydrogen sulfide. The whole genome RNA was extracted by the treated fission yeast, and after the reverse transcriptional gene chip hybridization. The signal was standardized by SAS analysis system, and 83 differentially expressed genes were screened by 2-fold and t-test analysis, and 280 differential genes were screened by 1.5-fo1d. Through cluster The analysis found that the three biological repeat experiments were repeatable and the experimental conclusions were reliable. The real-time quantitative PCR analysis of the 1.2-fold change gene found that the reliability of the gene chip data was high, so we used 1.5-fold as the main analysis of the genes.
DAVID online software was used to classify the differentially expressed genes by default (counts2, EASE score < 0.1). The 2-fold gene GO bioprocess classification showed that there were 20 down-regulation genes and 63 genes up regulation of.1.5 fold, 156 genes were up-regulated and 124 genes were downregulated. Among them, 31 genes in 2-fold and stress response Related, 18 genes encode the predicted or known transporters, 11 genes encode proteins associated with cell cycle / meiosis, and 10 genes encoded proteins participate in redox reaction.
Comparison with stress related genes found that 46% of the gene induced by hydrogen sulfide was the same as hydrogen peroxide, 43% of the gene was the same as cadmium, and 49% of the gene was the same as heat. However, only 12% and 24% hydrogen sulfide induced genes were the same as sorbitol and methyl methanosylsulfonate. Oxidative stress is involved in the expression of some genes in the MAPK signaling pathway, which may be the target gene of hydrogen sulfide.
In the whole genome protein localization study, 480 of the 4954 genes are located in the mitochondria (9.6% genes are located in the mitochondria). More than 1.5 times more than 124 genes are down, 23 of the genes in the mitochondria are down regulated by the hydrogen sulfide treatment (the 18.5% gene is located in the mitochondria). It shows that hydrogen sulfide causes many mitochondrial genes. Down regulation may have an effect on the function of mitochondria.
The effect of hydrogen sulfide on mitochondrial function was verified by detecting oxygen consumption and mitochondrial membrane potential in the respiration of fission yeast. The experimental results showed that the oxygen consumption of fission yeast after hydrogen sulfide treatment was reduced, the mitochondrial membrane potential decreased, which proved mitochondrial function damage, which fully confirmed the results of DNA chip data analysis, hydrogen sulfide caused by hydrogen sulfide. The expression of mitochondrial gene was downregulated.
Some gene differential expression induced by hydrogen sulfide can provide clues for human homologous protein research and molecular targets, providing a basis for further exploration of the mechanism and treatment of human disease related to hydrogen sulfide.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：Q78;R3416

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本文編號：2014838