銅綠假單胞菌las群體感應(yīng)系統(tǒng)反義肽核酸的構(gòu)建及其生物學(xué)作用研究
發(fā)布時間：2018-08-20 11:28
【摘要】：目的1.針對銅綠假單胞菌las群體感應(yīng)系統(tǒng)的lasR/lasI基因,設(shè)計和篩選出與其mRNA緊密結(jié)合的反義寡核苷核酸序列,以此為基礎(chǔ)設(shè)計合成反義肽核酸。2.評估反義肽核酸對銅綠假單胞菌PAO1的生長、lasR/lasI基因表達(dá)、生物被膜形成及毒力因子表達(dá)的影響。方法一、銅綠假單胞菌PAO1 lasR和lasI基因反義肽核酸的設(shè)計與篩選1.銅綠假單胞菌PAO1 lasR和lasI基因反義寡核苷酸的設(shè)計與篩選1.1目的基因的擴增:根據(jù)Gene Bank數(shù)據(jù)庫提供的銅綠假單胞菌PAO1 lasR和lasI全長基因序列,用軟件Primer Primer 6.0設(shè)計擴增引物,進行l(wèi)asR和lasI基因的PCR擴增。1.2構(gòu)建lasR和lasI基因mRNA表達(dá)質(zhì)粒:回收并純化lasR和lasI基因擴增產(chǎn)物,與質(zhì)粒載體p GEM-T easy vector進行連接重組。1.3重組質(zhì)粒p GEM-T easy vector-lasR/lasI的驗證:重組質(zhì)粒轉(zhuǎn)化感受態(tài)細(xì)菌DH5α,利用藍(lán)白斑實驗篩選出陽性克隆。擴增后提取p GEM-T easy vector-lasR/lasI質(zhì)粒并進行p GEM-T easy vector-lasR/lasI Not I酶切鑒定和測序鑒定。1.4利用軟件RNA structure 5.2設(shè)計lasR和lasI基因mRNA反義寡核苷酸序列。1.5反義寡核苷酸的篩選:進行l(wèi)asR/lasI mRNA體外轉(zhuǎn)錄,利用斑點雜交實驗篩選出反義寡核苷酸序列。2.銅綠假單胞菌PAO1 lasR和lasI基因反義肽核酸的設(shè)計與合成依據(jù)篩選出的反義寡核苷酸序列,按照肽核酸設(shè)計原則設(shè)計合成銅綠假單胞菌PAO1 lasR和lasI基因反義肽核酸,并將其與穿膜肽(KFF)3K連接。二、銅綠假單胞菌PAO1 lasR和lasI基因反義肽核酸生物學(xué)作用研究1.反義肽核酸對銅綠假單胞菌PAO1 lasR和lasI基因表達(dá)的影響:利用q PCR方法檢測lasR和lasI基因mRNA相對表達(dá)量。2.反義肽核酸對銅綠假單胞菌PAO1生長的影響:酶標(biāo)儀測定LB液體培養(yǎng)基中銅綠假單胞菌PAO1 OD600和LB固體培養(yǎng)基計數(shù)菌落。3.反義肽核酸對銅綠假單胞菌PAO1生物被膜形成的影響:光學(xué)顯微鏡、掃描電鏡和激光共聚焦顯微鏡觀察生物膜內(nèi)細(xì)菌含量。4.反義肽核酸對銅綠假單胞菌PAO1毒力因子表達(dá)的影響:采用ELISA技術(shù)檢測銅綠假單胞菌PAO1外毒素A、綠膿菌素含量的變化,q PCR方法檢測彈力蛋白酶lasB基因mRNA相對表達(dá)量。結(jié)果一、銅綠假單胞菌PAO1 lasR和lasI基因反義肽核酸的設(shè)計與篩選1.成功構(gòu)建lasR和lasI基因mRNA表達(dá)重組質(zhì)粒p GEM-T easy vector-lasR/lasI,篩選出與銅綠假單胞菌PAO1 lasR和lasI基因mRNA緊密結(jié)合的反義寡核苷酸序列。2.以篩選出的反義寡核苷酸序列為基礎(chǔ)合成反義肽核酸,并將其與穿膜肽結(jié)合構(gòu)建了穿膜肽-反義肽核酸。二、銅綠假單胞菌PAO1 lasR和lasI基因反義肽核酸生物學(xué)作用研究1.lasR/lasI穿膜肽-反義肽核酸抑制lasR/lasI mRNA的表達(dá),與對照組相比,其表達(dá)量有顯著差異,并且隨著穿膜肽-反義肽核酸濃度增加,對lasR/lasI mRNA的表達(dá)抑制作用增強。2.lasR/lasI穿膜肽-反義肽核酸濃度"g40μM時,對銅綠假單胞菌PAO1的生長有明顯抑制作用,濃度越高,對生長的抑制作用越明顯。而穿膜肽和反義肽核酸本身對細(xì)菌的生長無明顯抑制作用。3.光學(xué)顯微鏡、掃描電鏡和激光共聚焦顯微鏡觀察結(jié)果顯示,lasR/lasI穿膜肽-反義肽核酸轉(zhuǎn)染的銅綠假單胞菌PAO1,其生物被膜的形成明顯抑制。4.lasR/lasI調(diào)控的毒力因子彈性蛋白酶lasB、綠膿菌素和外毒素A等在穿膜肽-反義肽核酸的作用下表達(dá)量均下降,與陰性對照組相比,差異有統(tǒng)計學(xué)意義。結(jié)論1.通過體外表達(dá)和斑點雜交實驗,成功效篩選出與目的基因lasR/lasI mRNA緊密結(jié)合的反義寡核苷酸序列,并在此基礎(chǔ)上成功設(shè)計合成反義肽核酸并構(gòu)建了穿膜肽連接的穿膜肽-反義肽核酸。2.穿膜肽-反義肽核酸明顯抑制lasR/lasI mRNA的表達(dá),實現(xiàn)對銅綠假單胞菌las群體感應(yīng)系統(tǒng)的淬滅。3.lasR/lasI mRNA穿膜肽-反義肽核酸能夠顯著抑制銅綠假單胞菌PAO1生物被膜的形成。4.穿膜肽-反義肽核酸成功抑制銅綠假單胞菌PAO1的生長,并明顯下調(diào)毒力因子彈性蛋白酶B基因及綠膿菌素和外毒素A的表達(dá)。5.本研究設(shè)計的穿膜肽-反義肽核酸可通過對銅綠假單胞菌las群體感應(yīng)系統(tǒng)的淬滅,顯著抑制銅綠假單胞菌PAO1的生長及毒力相關(guān)因子的表達(dá),為銅綠假單胞菌感染的治療提供新的思路。
[Abstract]:Aim 1. To design and screen antisense oligonucleotide sequences that bind tightly to the Las quorum sensing system of Pseudomonas aeruginosa and synthesize antisense peptide nucleotides. 2. To evaluate the effect of antisense peptide nucleic acids on the growth, lasR/lasI gene expression, biofilm formation and virulence factors of Pseudomonas aeruginosa PAO 1. Methods 1. Design and screening of antisense oligonucleotides of PAO1 lasR and lasI genes in Pseudomonas aeruginosa 1. Design and screening of antisense oligonucleotides of PAO1 lasR and lasI genes in Pseudomonas aeruginosa 1.1. Amplification of target genes: According to the full-length gene sequences of PAO1 lasR and lasI in Pseudomonas aeruginosa provided by Gene Bank database, using software Pr IMER Primer 6.0 designed amplification primers to amplify lasR and lasI genes by PCR. 1.2 constructed lasR and lasI gene mRNA expression plasmids: recovered and purified lasR and lasI gene amplification products, and linked recombinant plasmid P GEM-T easy vector. 1.3 recombinant plasmid P GEM-T easy vector-lasR/lasI validation: recombinant plasmid transformed competent bacteria D GEM-T easy vector-lasI H5a, positive clones were screened out by blue-and-white spot assay. After amplification, plasmids of P GEM-T easy vector-lasR/lasI were extracted and identified by digestion and sequencing of P GEM-T easy vector-lasR/lasI Not I. 1.4 The antisense oligonucleotide sequences of lasR and lasI genes were designed by software RNA structure 5.2. 1.5 antisense oligonucleotides were screened: lasR/lasI antisense oligonucleotides were screened. I mRNA was transcribed in vitro and antisense oligonucleotide sequences were screened out by dot blot hybridization. 2. The antisense oligonucleotide sequences of PAO1 lasR and lasI genes in Pseudomonas aeruginosa were designed and synthesized according to the principle of peptide nucleic acid design and synthesis. Linking to KFF 3K. 2. Biological effects of antisense peptide nucleic acids of PAO1 lasR and lasI genes in Pseudomonas aeruginosa 1. Effects of antisense peptide nucleic acids on the expression of PAO1 lasR and lasI genes in Pseudomonas aeruginosa: The relative expression of lasR and lasI mRNA was detected by q-PCR. 2. Effects of antisense peptide nucleic acids on the growth of PAO1 in Pseudomonas aeruginosa. Enzyme-labeled assay was used to determine the number of colonies in the LB liquid medium of Pseudomonas aeruginosa PAO1 OD600 and LB solid medium. 3. Effect of antisense peptide nucleic acid on the biofilm formation of Pseudomonas aeruginosa PAO1: observation of bacterial content in biofilm by optical microscope, scanning electron microscope and laser confocal microscope. 4. Toxicity of antisense peptide nucleic acid to Pseudomonas aeruginosa PAO1 Effect of Force Factor Expression: ELISA was used to detect the content of exotoxin A and Pseudomonas aeruginosa PAO1, and q-PCR was used to detect the relative expression of elastase lasB gene mRNA. Results 1. Design and screening of antisense peptide nucleic acid of PAO1 lasR and lasI gene in Pseudomonas aeruginosa 1. The expression of lasR and lasI gene mRNA was constructed successfully. The antisense oligonucleotide sequence closely associated with PAO1 lasR and lasI gene mRNA of Pseudomonas aeruginosa was screened out by P GEM-T easy vector-lasR/lasI. 2. The antisense oligonucleotide was synthesized on the basis of the antisense oligonucleotide sequence and combined with the penetrating peptide to construct the penetrating peptide-antisense peptide nucleic acid. 2. Pseudomonas aeruginosa PAO1 Las Biological effects of antisense peptide nucleic acid of R and lasI gene 1. LasR/lasI penetrating peptide-antisense peptide nucleic acid inhibited the expression of lasR/lasI mRNA. Compared with the control group, the expression of lasR/lasI mRNA was significantly different, and the inhibitory effect on the expression of lasR/lasI mRNA was enhanced with the increasing concentration of penetrating peptide-antisense peptide nucleic acid. 2. LasR/lasI penetrating peptide-antisense peptide nucleic acid was concentrated. The higher the concentration, the more obvious the inhibitory effect on the growth of PAO1. But the transmembrane peptide and antisense peptide nucleic acid have no obvious inhibitory effect on the growth of bacteria. 3. The results of optical microscopy, scanning electron microscopy and laser confocal microscopy show that lasR / lasI transmembrane peptide-antisense peptide The expression of virulence factors, such as elastase lasB, Pseudomonas aeruginosa and exotoxin A, decreased under the action of transmembrane peptide-antisense peptide nucleic acid. Conclusion 1. There was significant difference between the negative control group and the transfected PAO1. The antisense oligonucleotide sequence closely bound to the target gene lasR/lasI mRNA was successfully screened out by hybridization experiment. On this basis, the antisense peptide nucleic acid was designed and synthesized successfully and the transmembrane peptide-antisense peptide nucleic acid was constructed. 2. The transmembrane peptide-antisense peptide nucleic acid significantly inhibited the expression of lasR/lasI mRNA and realized the antisense peptide nucleic acid against Pseudomonas aeruginosa. LasR/lasI mRNA penetrating peptide-antisense peptide nucleic acid can significantly inhibit the formation of biofilm of Pseudomonas aeruginosa PAO1. 4. penetrating peptide-antisense peptide nucleic acid successfully inhibits the growth of Pseudomonas aeruginosa PAO1, and significantly down-regulates the expression of virulence factor elastase B gene, Pseudomonas aeruginosa and exotoxin A. 5. This study was conducted to investigate the effect of lasR/lasI mRNA penetrating peptide-antisense peptide nucleic acid on the growth The designed penetrating peptide-antisense peptide nucleic acid can inhibit the growth of PAO1 and the expression of virulence-related factors by quenching the quorum sensing system of Pseudomonas aeruginosa las, which provides a new idea for the treatment of Pseudomonas aeruginosa infection.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R378
本文編號：2193422
[Abstract]:Aim 1. To design and screen antisense oligonucleotide sequences that bind tightly to the Las quorum sensing system of Pseudomonas aeruginosa and synthesize antisense peptide nucleotides. 2. To evaluate the effect of antisense peptide nucleic acids on the growth, lasR/lasI gene expression, biofilm formation and virulence factors of Pseudomonas aeruginosa PAO 1. Methods 1. Design and screening of antisense oligonucleotides of PAO1 lasR and lasI genes in Pseudomonas aeruginosa 1. Design and screening of antisense oligonucleotides of PAO1 lasR and lasI genes in Pseudomonas aeruginosa 1.1. Amplification of target genes: According to the full-length gene sequences of PAO1 lasR and lasI in Pseudomonas aeruginosa provided by Gene Bank database, using software Pr IMER Primer 6.0 designed amplification primers to amplify lasR and lasI genes by PCR. 1.2 constructed lasR and lasI gene mRNA expression plasmids: recovered and purified lasR and lasI gene amplification products, and linked recombinant plasmid P GEM-T easy vector. 1.3 recombinant plasmid P GEM-T easy vector-lasR/lasI validation: recombinant plasmid transformed competent bacteria D GEM-T easy vector-lasI H5a, positive clones were screened out by blue-and-white spot assay. After amplification, plasmids of P GEM-T easy vector-lasR/lasI were extracted and identified by digestion and sequencing of P GEM-T easy vector-lasR/lasI Not I. 1.4 The antisense oligonucleotide sequences of lasR and lasI genes were designed by software RNA structure 5.2. 1.5 antisense oligonucleotides were screened: lasR/lasI antisense oligonucleotides were screened. I mRNA was transcribed in vitro and antisense oligonucleotide sequences were screened out by dot blot hybridization. 2. The antisense oligonucleotide sequences of PAO1 lasR and lasI genes in Pseudomonas aeruginosa were designed and synthesized according to the principle of peptide nucleic acid design and synthesis. Linking to KFF 3K. 2. Biological effects of antisense peptide nucleic acids of PAO1 lasR and lasI genes in Pseudomonas aeruginosa 1. Effects of antisense peptide nucleic acids on the expression of PAO1 lasR and lasI genes in Pseudomonas aeruginosa: The relative expression of lasR and lasI mRNA was detected by q-PCR. 2. Effects of antisense peptide nucleic acids on the growth of PAO1 in Pseudomonas aeruginosa. Enzyme-labeled assay was used to determine the number of colonies in the LB liquid medium of Pseudomonas aeruginosa PAO1 OD600 and LB solid medium. 3. Effect of antisense peptide nucleic acid on the biofilm formation of Pseudomonas aeruginosa PAO1: observation of bacterial content in biofilm by optical microscope, scanning electron microscope and laser confocal microscope. 4. Toxicity of antisense peptide nucleic acid to Pseudomonas aeruginosa PAO1 Effect of Force Factor Expression: ELISA was used to detect the content of exotoxin A and Pseudomonas aeruginosa PAO1, and q-PCR was used to detect the relative expression of elastase lasB gene mRNA. Results 1. Design and screening of antisense peptide nucleic acid of PAO1 lasR and lasI gene in Pseudomonas aeruginosa 1. The expression of lasR and lasI gene mRNA was constructed successfully. The antisense oligonucleotide sequence closely associated with PAO1 lasR and lasI gene mRNA of Pseudomonas aeruginosa was screened out by P GEM-T easy vector-lasR/lasI. 2. The antisense oligonucleotide was synthesized on the basis of the antisense oligonucleotide sequence and combined with the penetrating peptide to construct the penetrating peptide-antisense peptide nucleic acid. 2. Pseudomonas aeruginosa PAO1 Las Biological effects of antisense peptide nucleic acid of R and lasI gene 1. LasR/lasI penetrating peptide-antisense peptide nucleic acid inhibited the expression of lasR/lasI mRNA. Compared with the control group, the expression of lasR/lasI mRNA was significantly different, and the inhibitory effect on the expression of lasR/lasI mRNA was enhanced with the increasing concentration of penetrating peptide-antisense peptide nucleic acid. 2. LasR/lasI penetrating peptide-antisense peptide nucleic acid was concentrated. The higher the concentration, the more obvious the inhibitory effect on the growth of PAO1. But the transmembrane peptide and antisense peptide nucleic acid have no obvious inhibitory effect on the growth of bacteria. 3. The results of optical microscopy, scanning electron microscopy and laser confocal microscopy show that lasR / lasI transmembrane peptide-antisense peptide The expression of virulence factors, such as elastase lasB, Pseudomonas aeruginosa and exotoxin A, decreased under the action of transmembrane peptide-antisense peptide nucleic acid. Conclusion 1. There was significant difference between the negative control group and the transfected PAO1. The antisense oligonucleotide sequence closely bound to the target gene lasR/lasI mRNA was successfully screened out by hybridization experiment. On this basis, the antisense peptide nucleic acid was designed and synthesized successfully and the transmembrane peptide-antisense peptide nucleic acid was constructed. 2. The transmembrane peptide-antisense peptide nucleic acid significantly inhibited the expression of lasR/lasI mRNA and realized the antisense peptide nucleic acid against Pseudomonas aeruginosa. LasR/lasI mRNA penetrating peptide-antisense peptide nucleic acid can significantly inhibit the formation of biofilm of Pseudomonas aeruginosa PAO1. 4. penetrating peptide-antisense peptide nucleic acid successfully inhibits the growth of Pseudomonas aeruginosa PAO1, and significantly down-regulates the expression of virulence factor elastase B gene, Pseudomonas aeruginosa and exotoxin A. 5. This study was conducted to investigate the effect of lasR/lasI mRNA penetrating peptide-antisense peptide nucleic acid on the growth The designed penetrating peptide-antisense peptide nucleic acid can inhibit the growth of PAO1 and the expression of virulence-related factors by quenching the quorum sensing system of Pseudomonas aeruginosa las, which provides a new idea for the treatment of Pseudomonas aeruginosa infection.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R378
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;2015年全國細(xì)菌耐藥監(jiān)測報告[J];中國執(zhí)業(yè)藥師;2016年03期
，本文編號：2193422
本文鏈接：http://sikaile.net/yixuelunwen/jichuyixue/2193422.html
最近更新
教材專著
