本文選題：抗菌肽 + 核酸作用機(jī)制��； 參考：《江南大學(xué)》2012年碩士論文

【摘要】：研究已證明,很多抗菌肽通過作用于細(xì)胞膜來起到抑菌作用,除此之外,也有研究表明很多抗菌肽能穿過細(xì)胞膜進(jìn)入胞內(nèi),通過作用于胞內(nèi)物質(zhì),擾亂細(xì)菌生理狀態(tài)而起到快速致命性的殺滅作用,但是關(guān)于抗菌肽對(duì)胞內(nèi)核酸的具體作用機(jī)制及作用于核酸后對(duì)細(xì)胞周期的影響研究很少。據(jù)此本文探究了抗菌肽BuforinⅡ的衍生物BuforinⅡ-A (BF2-A)和BuforinⅡ-B (BF2-B)對(duì)細(xì)胞膜,尤其是穿過細(xì)胞膜后對(duì)胞內(nèi)核酸的作用機(jī)制。 首先用凝膠阻滯實(shí)驗(yàn)觀察BF2-A和BF2-B與細(xì)菌DNA的結(jié)合。結(jié)果表明抗菌肽與金黃色葡萄球菌和大腸桿菌基因組DNA發(fā)生了結(jié)合。然后紫外光譜、圓二色譜和熒光光譜分別分析了這兩種抗菌肽與金黃色葡萄球菌/大腸桿菌基因組DNA結(jié)構(gòu)的影響及其結(jié)合方式。實(shí)驗(yàn)結(jié)果表明,加入肽后各譜圖中特征峰的強(qiáng)度發(fā)生了變化,但峰位幾乎沒有變化。這證明了BF2-A和BF2-B在體外分別與金黃色葡萄球菌和大腸桿菌基因組DNA發(fā)生了結(jié)合,使DNA結(jié)構(gòu)松散,但沒有破壞其雙螺旋結(jié)構(gòu); BF2-A和BF2-B均能與金黃色葡萄球菌和大腸桿菌基因組DNA結(jié)合并嵌入DNA堿基對(duì)中,且相同濃度的情況下這兩種肽與金黃色葡萄球菌基因組DNA的結(jié)合及嵌入程度均強(qiáng)于大腸桿菌基因組DNA,初步推測與金黃色葡萄球菌基因組DNA的AT含量大于大腸桿菌基因組DNA有關(guān)。此外,BF2-B對(duì)基因組DNA的作用效果強(qiáng)于BF2-A。 采用流式細(xì)胞術(shù)觀察了抗菌肽作用于菌后對(duì)其細(xì)胞膜結(jié)構(gòu)的影響。實(shí)驗(yàn)結(jié)果表明,在BF2-A和BF2-B的最低抑菌濃度時(shí),細(xì)菌細(xì)胞膜幾乎保持完整,尤其是BF2-A作用過的細(xì)菌。隨著BF2-A和BF2-B濃度的分別升高,細(xì)菌的細(xì)胞膜受到一定影響,但并沒有完全遭受破環(huán)。然后用凝膠阻滯電泳觀察抗菌肽作用于菌后對(duì)基因組DNA的影響,結(jié)果表明BF2-A和BF2-B進(jìn)入菌體內(nèi)都沒有引起細(xì)菌DNA的斷裂。然后用流式細(xì)胞術(shù)觀察抗菌肽作用于菌后對(duì)其細(xì)胞周期的影響,發(fā)現(xiàn)BF2-A和BF2-B將金黃色葡萄球菌和大腸桿菌抑制在細(xì)胞周期的DNA合成階段,此為BF2-A和BF2-B產(chǎn)生抑菌作用的一個(gè)重要原因。同時(shí),BF2-A和BF2-B對(duì)金黃色葡萄球菌DNA合成的抑制效果均好于大腸桿菌,再次說明這可能與金黃色葡萄球菌相關(guān)基因的AT含量大于大腸桿菌基因組DNA有關(guān)。同樣,上述各實(shí)驗(yàn)中,BF2-B對(duì)細(xì)菌細(xì)胞膜及胞內(nèi)的作用效果強(qiáng)于BF2-A。 用PCR技術(shù)獲得細(xì)菌DNA合成相關(guān)基因并通過凝膠阻滯實(shí)驗(yàn)驗(yàn)證抗菌肽與金黃色葡萄球菌和大腸桿菌DNA合成相關(guān)基因的結(jié)合。實(shí)驗(yàn)結(jié)果表明,BF2-A和BF2-B均能與DNA合成相關(guān)基因(DNA復(fù)制起始子基因、DNA聚合酶Ⅲ基因、DNA促旋酶基因等)結(jié)合,而且AT含量越高的DNA合成相關(guān)基因與BF2-A/BF2-B的結(jié)合強(qiáng)度越大。再一次證明BF2-A/BF2-B更容易與AT含量高的基因結(jié)合。此為金黃色葡萄球菌和大腸桿菌被抑制在DNA合成階段的一個(gè)重要原因。同時(shí),BF2-B對(duì)DNA合成相關(guān)基因的作用效果強(qiáng)于BF2-A。 實(shí)驗(yàn)結(jié)果說明,BF2-A/BF2-B能在對(duì)細(xì)菌細(xì)胞膜只產(chǎn)生十分微小的破環(huán)作用的情況下穿過細(xì)胞膜,在胞內(nèi)不斷裂DNA,卻能與DNA合成相關(guān)基因結(jié)合,而且DNA合成相關(guān)基因的AT含量越高,BF2-A/BF2-B與之結(jié)合的強(qiáng)度越大,最終將金黃色葡萄球菌和大腸桿菌的細(xì)胞周期抑制在DNA合成階段。此外,從所有的實(shí)驗(yàn)中都可以看出,BF2-B的作用效果好于BF2-A,這是BF2-B的抑菌效果好于BF2-A的重要原因。
[Abstract]:Studies have shown that many antimicrobial peptides by acting on the cell membrane plays an inhibitory effect, in addition, studies have also shown that many antimicrobial peptides can cross the cell membrane into the intracellular acting through intracellular substances, bacteria and disrupt the physiological state to kill rapidly fatal, but the specific mechanism of antibacterial peptide on intracellular nucleic acid and nucleic acid in after effects on cell cycle. This paper explores the little research of antibacterial peptide Buforin II derivatives of Buforin II -A (BF2-A) and Buforin -B (BF2-B) on the cell membrane, especially the mechanism of intracellular nucleic acids across the cell membrane.
First, combined with gel retardation experiments to observe the BF2-A and BF2-B and DNA of bacteria. The results showed that the antibacterial peptide with Staphylococcus aureus and Escherichia coli DNA genome occurred. Then combined with UV spectroscopy, chromatography and fluorescence spectra of round two respectively and analyzes the two kinds of antibacterial peptide and Staphylococcus aureus Escherichia coli genome structure of DNA / influence and their combination. The experimental results show that the change of spectrum characteristic peak after the addition of peptide in strength, but almost no change in the peak position. It proved that BF2-A and BF2-B in vitro respectively with Staphylococcus aureus and Escherichia coli DNA genome occurred with DNA, the structure is loose, but no damage to the double spiral structure; BF2-A and BF2-B and Staphylococcus aureus and Escherichia coli genomic DNA binding and insertion of DNA base pairs, and the case of the same concentration of the two peptides with Staphylococcus aureus The binding and embedding degree of genomic DNA is stronger than that of Escherichia coli genome DNA. It is preliminarily speculated that the AT content of genomic DNA of Staphylococcus aureus is larger than that of Escherichia coli genome DNA. In addition, the effect of BF2-B on genomic DNA is stronger than BF2-A..
The observation of antibacterial peptides in bacteria on the cell membrane structure of flow cytometry. The experimental results show that the minimum inhibitory concentration of BF2-A and BF2-B, the bacterial cell membrane remained almost intact, especially BF2-A treated bacteria. With BF2-A and BF2-B concentration were increased, the bacterial cell membrane have a certain influence, but not completely damage. Then by gel electrophoresis were used to observe the effect of antimicrobial peptides to block bacteria on genomic DNA, the results showed that BF2-A and BF2-B in bacteria in vivo did not cause rupture of bacterial DNA. Then by flow cytometry on antimicrobial peptides on the cell cycle of bacteria effect of DNA, BF2-A and BF2-B found that the synthesis stage to Staphylococcus aureus and Escherichia coli in the inhibition of the cell cycle, an important reason for this is BF2-A and BF2-B have inhibitory effect. At the same time, BF2-A and BF2-B of golden color The inhibitory effect of Staphylococcus aureus DNA synthesis is better than that of Escherichia coli, which again indicates that this may be related to the AT content of Staphylococcus aureus related genes larger than that of Escherichia coli genomic DNA. Similarly, in these experiments, BF2-B has stronger effect on bacterial cell membrane and intracellular than BF2-A..
A combination of bacterial DNA synthesis related genes and antimicrobial peptides with Staphylococcus aureus and Escherichia coli DNA synthesis related genes by gel retardation experiments using PCR technology. The experimental results show that BF2-A and BF2-B can DNA synthesis related genes (DNA gene replication, DNA polymerase III gene, DNA gyrase gene etc.) with the content of AT is higher and the bonding strength of the DNA synthesis related genes and BF2-A/BF2-B more. Once again proved BF2-A/BF2-B more easily with high AT content. This is the combination of genes of Staphylococcus aureus and Escherichia coli were inhibited in one of the important reasons of DNA synthesis phase. At the same time, the effect of BF2-B on DNA synthesis the gene has a better effect than BF2-A.
The experimental results show that BF2-A/BF2-B can produce only on bacterial cell membrane of very small broken ring under conditions across the cell membrane, the intracellular DNA does not break, but can be combined with DNA synthesis related genes, the content of AT and DNA synthesis related genes with higher BF2-A/BF2-B with greater strength, will eventually the cell cycle of Staphylococcus aureus and Escherichia coli in inhibition of DNA synthesis stage. In addition, from all experiments we can see that the effect of BF2-B is better than BF2-A, which is an important reason for the inhibitory effect of BF2-B is better than BF2-A.

【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R151

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 皮新春,鄒國林;限制性核酸內(nèi)切酶與DNA相互作用研究進(jìn)展[J];氨基酸和生物資源;1997年02期

2 杜江燕,王炳祥,徐飛,陳婷婷,沈珠英,沈永淼,邢巍,陸天虹;1,4-二氫吡啶衍生物與DNA相互作用的電化學(xué)與圓二色譜研究[J];高等學(xué)校化學(xué)學(xué)報(bào);2005年04期

3 楊源源;張志超;盛輝;劉鳳玉;錢旭紅;徐芹;張晶;;一種苊并雜環(huán)有機(jī)小分子嵌入DNA的幾何學(xué)模式研究[J];高等學(xué)�；瘜W(xué)學(xué)報(bào);2007年03期

4 劉雪平;馮素玲;潘自紅;樊靜;;溴化乙錠探針研究勞氏青蓮與脫氧核糖核酸的相互作用[J];光譜學(xué)與光譜分析;2006年10期

5 曾戎;劉宏偉;趙劍豪;屠美;;磷酰化殼聚糖仿生衍生物的合成及其與DNA的相互作用[J];暨南大學(xué)學(xué)報(bào)(自然科學(xué)版);2008年01期

6 王鑫，劉丕旭，，章靜波，薛社普;紅細(xì)胞終末分化期出現(xiàn)與珠蛋白基因表達(dá)增強(qiáng)子HS2序列特異結(jié)合的蛋白質(zhì)因子[J];解剖學(xué)報(bào);1994年04期

7 那淑敏,賈士芳,陳秀珠,陳美玲,還連棟,郭興華,李金波,李金燕,劉軍;嗜酸乳桿菌產(chǎn)生的廣譜抗菌肽AP311的分離和鑒定[J];微生物學(xué)報(bào);2001年04期

8 郝剛;施用暉;唐亞麗;樂國偉;;抗菌肽BuforinⅡ衍生物與大腸桿菌基因組DNA的作用機(jī)制[J];微生物學(xué)報(bào);2010年03期

9 王關(guān)林;唐金花;蔣丹;方宏筠;劉照斌;;苦參對(duì)雞大腸桿菌的抑菌作用及其機(jī)理研究[J];中國農(nóng)業(yè)科學(xué);2006年05期

10 魯開國,胡智慧,生菊,陶秀蓮,王偉棟,王慧;PCR檢測副溶血弧菌耐熱直接溶血素和耐熱直接相關(guān)溶血素基因的研究[J];中國衛(wèi)生檢驗(yàn)雜志;1995年02期

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