铜绿假单胞菌las群体感应系统反义肽核酸的构建及其生物学作用研究
发布时间：2018-08-20 11:28
【摘要】：目的1.针对铜绿假单胞菌las群体感应系统的lasR/lasI基因,设计和筛选出与其mRNA紧密结合的反义寡核苷核酸序列,以此为基础设计合成反义肽核酸。2.评估反义肽核酸对铜绿假单胞菌PAO1的生长、lasR/lasI基因表达、生物被膜形成及毒力因子表达的影响。方法一、铜绿假单胞菌PAO1 lasR和lasI基因反义肽核酸的设计与筛选1.铜绿假单胞菌PAO1 lasR和lasI基因反义寡核苷酸的设计与筛选1.1目的基因的扩增:根据Gene Bank数据库提供的铜绿假单胞菌PAO1 lasR和lasI全长基因序列,用软件Primer Primer 6.0设计扩增引物,进行lasR和lasI基因的PCR扩增。1.2构建lasR和lasI基因mRNA表达质粒:回收并纯化lasR和lasI基因扩增产物,与质粒载体p GEM-T easy vector进行连接重组。1.3重组质粒p GEM-T easy vector-lasR/lasI的验证:重组质粒转化感受态细菌DH5α,利用蓝白斑实验筛选出阳性克隆。扩增后提取p GEM-T easy vector-lasR/lasI质粒并进行p GEM-T easy vector-lasR/lasI Not I酶切鉴定和测序鉴定。1.4利用软件RNA structure 5.2设计lasR和lasI基因mRNA反义寡核苷酸序列。1.5反义寡核苷酸的筛选:进行lasR/lasI mRNA体外转录,利用斑点杂交实验筛选出反义寡核苷酸序列。2.铜绿假单胞菌PAO1 lasR和lasI基因反义肽核酸的设计与合成依据筛选出的反义寡核苷酸序列,按照肽核酸设计原则设计合成铜绿假单胞菌PAO1 lasR和lasI基因反义肽核酸,并将其与穿膜肽(KFF)3K连接。二、铜绿假单胞菌PAO1 lasR和lasI基因反义肽核酸生物学作用研究1.反义肽核酸对铜绿假单胞菌PAO1 lasR和lasI基因表达的影响:利用q PCR方法检测lasR和lasI基因mRNA相对表达量。2.反义肽核酸对铜绿假单胞菌PAO1生长的影响:酶标仪测定LB液体培养基中铜绿假单胞菌PAO1 OD600和LB固体培养基计数菌落。3.反义肽核酸对铜绿假单胞菌PAO1生物被膜形成的影响:光学显微镜、扫描电镜和激光共聚焦显微镜观察生物膜内细菌含量。4.反义肽核酸对铜绿假单胞菌PAO1毒力因子表达的影响:采用ELISA技术检测铜绿假单胞菌PAO1外毒素A、绿脓菌素含量的变化,q PCR方法检测弹力蛋白酶lasB基因mRNA相对表达量。结果一、铜绿假单胞菌PAO1 lasR和lasI基因反义肽核酸的设计与筛选1.成功构建lasR和lasI基因mRNA表达重组质粒p GEM-T easy vector-lasR/lasI,筛选出与铜绿假单胞菌PAO1 lasR和lasI基因mRNA紧密结合的反义寡核苷酸序列。2.以筛选出的反义寡核苷酸序列为基础合成反义肽核酸,并将其与穿膜肽结合构建了穿膜肽-反义肽核酸。二、铜绿假单胞菌PAO1 lasR和lasI基因反义肽核酸生物学作用研究1.lasR/lasI穿膜肽-反义肽核酸抑制lasR/lasI mRNA的表达,与对照组相比,其表达量有显著差异,并且随着穿膜肽-反义肽核酸浓度增加,对lasR/lasI mRNA的表达抑制作用增强。2.lasR/lasI穿膜肽-反义肽核酸浓度"g40μM时,对铜绿假单胞菌PAO1的生长有明显抑制作用,浓度越高,对生长的抑制作用越明显。而穿膜肽和反义肽核酸本身对细菌的生长无明显抑制作用。3.光学显微镜、扫描电镜和激光共聚焦显微镜观察结果显示,lasR/lasI穿膜肽-反义肽核酸转染的铜绿假单胞菌PAO1,其生物被膜的形成明显抑制。4.lasR/lasI调控的毒力因子弹性蛋白酶lasB、绿脓菌素和外毒素A等在穿膜肽-反义肽核酸的作用下表达量均下降,与阴性对照组相比,差异有统计学意义。结论1.通过体外表达和斑点杂交实验,成功效筛选出与目的基因lasR/lasI mRNA紧密结合的反义寡核苷酸序列,并在此基础上成功设计合成反义肽核酸并构建了穿膜肽连接的穿膜肽-反义肽核酸。2.穿膜肽-反义肽核酸明显抑制lasR/lasI mRNA的表达,实现对铜绿假单胞菌las群体感应系统的淬灭。3.lasR/lasI mRNA穿膜肽-反义肽核酸能够显著抑制铜绿假单胞菌PAO1生物被膜的形成。4.穿膜肽-反义肽核酸成功抑制铜绿假单胞菌PAO1的生长,并明显下调毒力因子弹性蛋白酶B基因及绿脓菌素和外毒素A的表达。5.本研究设计的穿膜肽-反义肽核酸可通过对铜绿假单胞菌las群体感应系统的淬灭,显著抑制铜绿假单胞菌PAO1的生长及毒力相关因子的表达,为铜绿假单胞菌感染的治疗提供新的思路。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
本文编号：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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
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1 ;2015年全国细菌耐药监测报告[J];中国执业药师;2016年03期
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