致犊牛脑膜炎大肠杆菌ibeB基因缺失株的构建及部分生物学特性研究

发布时间：2018-05-03 05:04

本文选题：肠外致病性大肠杆菌 + ibeB基因缺失株　；参考：《石河子大学》2017年硕士论文

【摘要】：肠外致病性大肠杆菌(Extraintestinal Pathogenic E.coli,Ex PEC),是一类新的致病性大肠杆菌菌群,定植于宿主肠道外其它组织,主要导致肠道外的感染。该菌可穿越人和动物肠屏障及血脑屏障,在临床上能够引起新生儿脑膜炎和尿路感染;犊牛的腹泻、脑膜炎和败血症等,临床发病率与病死率都较高。Ex PEC能引起中枢系统疾病,多因素参与其致病过程,尤其是侵袭素Ibe家族蛋白Ibe A、Ibe B、Ibe C等毒力因子在致新生儿脑膜炎大肠杆菌K1株黏附、侵袭宿主细胞中扮演重要角色。前期本实验室从神经症状的犊牛脑组织中分离到血清型为O161型的E.coli-SN株,侵袭素Ibe家族蛋白在E.coli-SN株是否存在,与致新生儿脑膜炎大肠杆菌K1株有什么样的关系,是否参与E.coli-SN株的致病性,目前尚不清楚。本研究以临床犊牛脑炎分离株E.coli-SN为研究对象,采用PCR方法检测ibe B基因,通过Red/ET同源重组系统构建ibe B基因缺失突变株E.coli-SN-Δibe B,比较亲本株与突变株之间在体外生物学特性和小鼠致病性的差异,为Ex PEC致病机制奠定基础。主要研究内容和结果如下:1.致犊牛脑炎大肠杆菌分离株ibe B基因的序列分析:为了分析致犊牛脑膜炎大肠杆菌分离株ibe B基因的序列。采用PCR方法,从分菌株克隆ibe B基因,连接到p MD19-T构成p MD19-T-ibe B,经测序、拼接、比对后获得ibe B基因全长序列,并采用DNAStar软件分析分菌株ibe B基因序列。试验结果表明:E.coli-SN株与标准株大肠杆菌K1 RS218核苷酸和氨基酸同源性分别90.5%和96.9%,进化树聚为一支;E.coli-SG株与大肠杆菌K12的核苷酸和氨基酸同源性分别为99.4%和100.0%,进化树聚为一支。初步揭示致犊牛脑膜炎大肠杆菌分离株E.coli-SN与致新生儿脑膜炎大肠杆菌K1株ibe B基因密切相关,亲缘性更近。2.E.coli-SN ibe B基因缺失株的构建及鉴定:为深入研究ibe B基因在E.coli-SN致病力中的作用,本研究应用Red/ET同源重组技术对目的基因ibe B进行了敲除。根据目的基因ibe B的上下游序列和FRT-PGK-gb2-kan/neo-FRT序列设计一对引物用于同源重组缺失株的构建。第一步将质粒p Red ET电转化入E.coli-SN的感受态细胞中,30℃培养;L-阿拉伯糖诱导质粒p Red ET的表达。第二步电转化,将FPF-ibe B同源臂片段电转化入E.coli-SN-p Red ET感受态细胞中。第三步将707-FLPe质粒电转化入E.coli-SN-Δibe B-FPF感受态细胞中,37℃诱导表达,将标记性基因丢失,所有重组菌株均采用相应PCR验证鉴定。37℃、无抗性压力条件下传代培养20代,PCR检测无返祖现象,表明突变菌株具有良好的遗传稳定性。即成功构建E.coli-SN基因缺失株E.coli-SN-Δibe B。3.E.coli-SN-Δibe B基因缺失株部分生物学特性的研究:为了明确E.coli-SN ibe B基因的功能,本试验进行了缺失突变株的溶血试验,体外耐酸耐碱培养试验,毒力检测及对小鼠肝、脑、脾载菌量的测定。结果显示:突变株的溶血特性、体外耐酸耐碱并未发生改变;与亲本株E.coli-SN相比较,E.coli-SN-?ibe B突变株对小鼠LD50高出101.16倍,但差异不显著;接种突变株E.coli-SN-?ibe B在小鼠肝组织载菌量均显著降低(t=24h,p0.01;t=48h,p0.05),在脾脏组织的载菌量也均显著降低(t=24h,p0.05;t=48h,p0.01),但在脑组织中的载菌量减少但不显著;感染E.coli-SN-?ibe B缺失株小鼠存活时间(平均3.38天)比E.coli-SN亲本株(平均2.25天)显著延长(p0.05)。结果表明ibe B毒力基因的缺失可减弱E.coli-SN菌株的毒力。
[Abstract]:Extraintestinal Pathogenic E.coli (Ex PEC), a new pathogenic Escherichia coli group, is a new group of pathogenic Escherichia coli colonized in other tissues outside the host intestinal tract, which mainly causes intestinal infection. This bacterium can pass through human and animal intestinal barrier and blood brain barrier, and can cause neonatal meningitis and urinary tract infection on the bed; calves Diarrhea, meningitis and septicaemia, the clinical morbidity and mortality are higher.Ex PEC can cause central system disease, multiple factors participate in its pathogenesis, especially the Ibe family protein Ibe A, Ibe B, Ibe C and other virulence factors in the neonatal meningitis Escherichia coli K1 strain to play an important role in the invasion of host cells. The laboratory has isolated the serotype O161 type E.coli-SN from the neurotic calf brain tissue, the presence of the Ibe family protein of the invasive element in the E.coli-SN strain, the relationship with the K1 strain of the neonatal meningitis Escherichia coli and the pathogenicity of the E.coli-SN strain, is not clear. This study is based on clinical calf encephalitis isolate. E.coli-SN was used as the research object to detect IBE B gene by PCR method and to construct IBE B gene deletion mutant E.coli-SN- delta IBE B by Red/ET homologous recombination system. The difference of biological characteristics and pathogenicity in vitro between parental and mutant strains was compared to establish the basis for Ex PEC pathogenesis. The main contents and results are as follows: 1. calves Sequence analysis of IBE B gene of bovine encephalitis Escherichia coli isolate: in order to analyze the sequence of IBE B gene of calf meningitis Escherichia coli isolate strain, the PCR method was used to clone the IBE B gene from the isolates to P MD19-T to form P MD19-T-ibe B, and the sequence was sequenced and compared. The IBE B gene sequence of the strain showed that the E.coli-SN and the amino acids of the standard strain of Escherichia coli K1 RS218 were 90.5% and 96.9%, respectively, and the evolutionary tree was one branch; the nucleotide and amino acids of the E.coli-SG and Escherichia coli K12 were 99.4% and 100%, respectively, and the evolutionary tree was one. E.coli-SN, an inflammatory Escherichia coli isolate, is closely related to the IBE B gene of neonatal meningitis Escherichia coli K1 strain, and its affinity is more closely related to the construction and identification of the.2.E.coli-SN IBE B gene deletion strain. The purpose of this study is to investigate the role of IBE B gene in E.coli-SN pathogenicity. This study uses Red/ET Co source recombination technology to knock off the target gene IBE. Design a pair of primers based on the upstream and downstream sequences of the target gene IBE B and the FRT-PGK-gb2-kan/neo-FRT sequence for the construction of the homologous recombination deletion strain. The first step was to convert the plasmid P Red ET into E.coli-SN receptive cells, at 30 C, and the P Red ET of L- Arabia sugar induced plasmid. The second step electrical transformation was used to convert the FPF-ibe to homologous arms. The segment electricity was converted into the E.coli-SN-p Red ET receptive cells. The 707-FLPe plasmid was converted into the E.coli-SN- delta IBE B-FPF receptive cells by third steps. The expression was induced at 37 degrees C, and the marker genes were lost. All the recombinant strains were identified by the corresponding PCR verification at.37, 20 generations under no resistant pressure strips, and PCR was used to detect no reversion The mutation strains have good genetic stability. That is, a successful construction of the partial biological characteristics of the deletion strain of the E.coli-SN gene E.coli-SN- delta IBE B.3.E.coli-SN- delta IBE B gene. In order to clarify the function of the E.coli-SN IBE B gene, the experiment carried out the hemolytic test of the missing mutant strain, the acid resistance and alkali resistance culture test in vitro, and the virulence. The results showed that the hemolytic characteristics of the mutant strain, the acid resistance and alkali resistance in vitro did not change, and the E.coli-SN-? IBE B mutant was 101.16 times higher than that of the parent strain E.coli-SN, but the difference was not significant; the inoculated mutant E.coli-SN-? IBE B in the liver tissues of the mice decreased significantly. Low (t=24h, P0.01; t=48h, P0.05) also significantly decreased the amount of bacteria carrying in the spleen (t=24h, P0.05; t=48h, P0.01), but decreased but not significant in the brain tissue; the survival time of the mice infected with E.coli-SN-? IBE B (average 3.38 days) was significantly longer than that of the E.coli-SN parent (average 2.25 days). The toxicity of E.coli-SN strain was weakened.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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