布鲁氏菌wbkC影响RAW264.7细胞基因表达的初步研究

发布时间：2018-02-04 18:22

本文关键词： 布鲁氏菌 B.melitensis M5-90-△wbkC菌株 miRNAs qRT-PCR 靶基因预测　出处：《海南大学》2017年硕士论文　论文类型：学位论文

【摘要】：布鲁氏菌(Brucela.melitensis)是革兰氏阴性菌,该病引起人和动物感染,在全世界较广泛流传,对人身安全构成威胁,给世界经济带来负担。wbkC作用于O-侧链的生物合成途径中催化GDP-4-NH2-4,6双脱氧甘露糖转换成GDP-4-甲酰胺基-4,6双脱氧甘露糖,是布鲁氏菌脂多糖O抗原合成的重要步骤。构建羊布鲁氏菌B.melitensis M5-90-△wbkC株,为研究与wbkC基因相关的microRNAs(miRNAs)奠定基础,为深入研究布鲁氏菌分子致病机制和设计更安全、更有效疫苗提供依据。本实验根据NCBI上B.melitensis M5-90基因组序列信息,查找wbkC基因序列及其上游(N)、下游(C)序列信息,查找pEGFP-N1质粒Kanar序列信息。分别设计wbkC基因N端C端同源臂和Kanar片段上下游引物。以B.melitensisM5-90基因组为模板,聚合酶链式反应(Polymerase Chain Reaction PCR)扩增wbkC基因左右同源臂序列。以pEGFP-N1质粒为模板PCR扩增Kanar序列。凝胶回收片段,分别连接至pMD20-T载体上。XbwI、KpnI酶切下N端同源臂,Kpnl、BamHI酶切下Kanar片段,BamHI、BstXI酶切下C端同源臂,回收片段。将pGEM-7ZF质粒也用相同的酶切,使产生相同的粘性末端,依次将N端同源臂、Kanar片段和C端同源臂连接pGEM-7ZF质粒,构建pGEM-7ZF-AwbkC自杀质粒,测序鉴定,将测序鉴定正确的pGEM-7ZF-△wbkC自杀质粒电转化至B.melitensis M5-90感受态细胞中,用含氨苄和卡那霉素双抗性选择培养基筛选阳性菌,菌落PCR鉴定,挑选的阳性菌培养使之同源重组,获得稳定遗传。成功构建B.melitensisM5-90-△wbkC 菌株。B.melitensis M5-90和B.melitens s M5-90-△wbkC 菌液,分别感染 RAW264.7 细胞,4 h后提取总RNA,分别进行miRNAs基因芯片分析,对B.melitensisM5-90和B.melitensis M5-90-AwbkC 在 RAW264.7 细胞差异表达 miRNAs,用 TaqMan 探针法实时荧光定量PCR(Quantitative Real-time PCR)对鉴定差异表达的miRNAs,验证结果:有10 miRNAs表达上下调结果和miRNAs基因芯片的热图结果相符合,其中,mmu-miR-574-3p、mmu-miR-669c-3p、mmu-miR-669p-3p、mmu-miR-466h-5p 表达下调,mmu-miR-1949、mmu-miR-155-5p、mmu-miR-6240、mmu-miR-3473a、mmu-miR-7012-5p、mmu-miR-7024-5p表达上调。利用在线网站对△△Ct2的5个miRNAs靶基因进行预测。
[Abstract]:Brucela.melitensis (Brucella melitensis) is a gram-negative bacterium that causes human and animal infections and is widely spread around the world and poses a threat to personal safety. The effect of wbkC on the biosynthesis of O- side chain catalyzes the conversion of GDP-4-NH _ 2-4N _ 6 dideoxymannose to GDP-4-formamide _ 4. 6 dideoxymannose is an important step in the synthesis of lipopolysaccharide O antigen of Brucella vulgaris. B.melitensis M5-90- wbkC strain was constructed. It lays a foundation for the study of microRNAsmiRNAsassociated with wbkC gene, and makes it safer to study the molecular pathogenic mechanism and design of Brucella. Based on the genome sequence information of B.melitensis M5-90 on NCBI, the wbkC gene sequence and its upstream sequence were searched. Downstream C) sequence information. To find out the Kanar sequence information of pEGFP-N1 plasmid. To design the homologous arm of N-terminal C terminal of wbkC gene and the upstream and downstream primers of Kanar fragment, respectively. 0 genome was used as template. Polymerase Chain Reaction PCR. The left and right arm sequences of wbkC gene were amplified. PEGFP-N1 plasmid was used as template PCR to amplify Kanar sequence. It was ligated to pMD20-T vector. XbwI- KpnI enzyme was used to cut down the Kanar fragment of KpnlBamHI. The pGEM-7ZF plasmid was digested with the same enzyme to produce the same sticky end, and then the N-terminal homologous arm was sequenced. Kanar fragment and C-terminal homologous arm were ligated with pGEM-7ZF plasmid to construct pGEM-7ZF-AwbkC suicide plasmid and sequenced. The correct suicide plasmid pGEM-7ZF- wbkC was transformed into B. melitensis M5-90 cells. Positive bacteria were screened by double resistant culture medium containing ampicillin and kanamycin, colony PCR was identified, and the selected positive bacteria were cultured for homologous recombination. Successful construction of B.melitensis M5-90- wbkC strains. B.melitensis M5-90 and B.melitens s. M5-90-wbkC liquid. The total RNAs were extracted from RAW264.7 cells for 4 h and were analyzed by miRNAs microarray. B. melitensis M5-90 and B.melitensis M5-90-AwbkC expressed miRNAs differently in RAW264.7 cells. PCR(Quantitative Real-time PCR was used to identify the differentially expressed miRNAs by TaqMan probe method. The results showed that 10 miRNAs down-regulated results were consistent with those of miRNAs microarray, including mmu-miR-574-3p. The expression of mmu-miR-669c-3pmmu-miR-669p-3pmmu-miR-466h-5p was down-regulated by mmu-miR-1949. Mmu-miR-155-5pmmu-miR-6240 mmu-miR-3473a mmu-miR-7012-5p. The expression of mmu-miR-7024-5p was up-regulated. Five miRNAs target genes of Ct2 were predicted by online website.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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