本文選題：腸外致病性大腸桿菌 + ibeB基因缺失株��； 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：腸外致病性大腸桿菌(Extraintestinal Pathogenic E.coli,Ex PEC),是一類新的致病性大腸桿菌菌群,定植于宿主腸道外其它組織,主要導(dǎo)致腸道外的感染。該菌可穿越人和動物腸屏障及血腦屏障,在臨床上能夠引起新生兒腦膜炎和尿路感染;犢牛的腹瀉、腦膜炎和敗血癥等,臨床發(fā)病率與病死率都較高。Ex PEC能引起中樞系統(tǒng)疾病,多因素參與其致病過程,尤其是侵襲素Ibe家族蛋白Ibe A、Ibe B、Ibe C等毒力因子在致新生兒腦膜炎大腸桿菌K1株黏附、侵襲宿主細胞中扮演重要角色。前期本實驗室從神經(jīng)癥狀的犢牛腦組織中分離到血清型為O161型的E.coli-SN株,侵襲素Ibe家族蛋白在E.coli-SN株是否存在,與致新生兒腦膜炎大腸桿菌K1株有什么樣的關(guān)系,是否參與E.coli-SN株的致病性,目前尚不清楚。本研究以臨床犢牛腦炎分離株E.coli-SN為研究對象,采用PCR方法檢測ibe B基因,通過Red/ET同源重組系統(tǒng)構(gòu)建ibe B基因缺失突變株E.coli-SN-Δibe B,比較親本株與突變株之間在體外生物學(xué)特性和小鼠致病性的差異,為Ex PEC致病機制奠定基礎(chǔ)。主要研究內(nèi)容和結(jié)果如下:1.致犢牛腦炎大腸桿菌分離株ibe B基因的序列分析:為了分析致犢牛腦膜炎大腸桿菌分離株ibe B基因的序列。采用PCR方法,從分菌株克隆ibe B基因,連接到p MD19-T構(gòu)成p MD19-T-ibe B,經(jīng)測序、拼接、比對后獲得ibe B基因全長序列,并采用DNAStar軟件分析分菌株ibe B基因序列。試驗結(jié)果表明:E.coli-SN株與標(biāo)準(zhǔn)株大腸桿菌K1 RS218核苷酸和氨基酸同源性分別90.5%和96.9%,進化樹聚為一支;E.coli-SG株與大腸桿菌K12的核苷酸和氨基酸同源性分別為99.4%和100.0%,進化樹聚為一支。初步揭示致犢牛腦膜炎大腸桿菌分離株E.coli-SN與致新生兒腦膜炎大腸桿菌K1株ibe B基因密切相關(guān),親緣性更近。2.E.coli-SN ibe B基因缺失株的構(gòu)建及鑒定:為深入研究ibe B基因在E.coli-SN致病力中的作用,本研究應(yīng)用Red/ET同源重組技術(shù)對目的基因ibe B進行了敲除。根據(jù)目的基因ibe B的上下游序列和FRT-PGK-gb2-kan/neo-FRT序列設(shè)計一對引物用于同源重組缺失株的構(gòu)建。第一步將質(zhì)粒p Red ET電轉(zhuǎn)化入E.coli-SN的感受態(tài)細胞中,30℃培養(yǎng);L-阿拉伯糖誘導(dǎo)質(zhì)粒p Red ET的表達。第二步電轉(zhuǎn)化,將FPF-ibe B同源臂片段電轉(zhuǎn)化入E.coli-SN-p Red ET感受態(tài)細胞中。第三步將707-FLPe質(zhì)粒電轉(zhuǎn)化入E.coli-SN-Δibe B-FPF感受態(tài)細胞中,37℃誘導(dǎo)表達,將標(biāo)記性基因丟失,所有重組菌株均采用相應(yīng)PCR驗證鑒定。37℃、無抗性壓力條件下傳代培養(yǎng)20代,PCR檢測無返祖現(xiàn)象,表明突變菌株具有良好的遺傳穩(wěn)定性。即成功構(gòu)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學(xué)特性的研究:為了明確E.coli-SN ibe B基因的功能,本試驗進行了缺失突變株的溶血試驗,體外耐酸耐堿培養(yǎng)試驗,毒力檢測及對小鼠肝、腦、脾載菌量的測定。結(jié)果顯示:突變株的溶血特性、體外耐酸耐堿并未發(fā)生改變;與親本株E.coli-SN相比較,E.coli-SN-?ibe B突變株對小鼠LD50高出101.16倍,但差異不顯著;接種突變株E.coli-SN-?ibe B在小鼠肝組織載菌量均顯著降低(t=24h,p0.01;t=48h,p0.05),在脾臟組織的載菌量也均顯著降低(t=24h,p0.05;t=48h,p0.01),但在腦組織中的載菌量減少但不顯著;感染E.coli-SN-?ibe B缺失株小鼠存活時間(平均3.38天)比E.coli-SN親本株(平均2.25天)顯著延長(p0.05)。結(jié)果表明ibe B毒力基因的缺失可減弱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.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61

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