本文選題：腸外致病性大腸桿菌大腸桿菌 + 比較基因組��； 參考：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：腸外致病性大腸桿菌(Extraintestinal pathogenic Escherichia coli,ExPEC)是引起人和動(dòng)物腸道外嚴(yán)重感染的病原體,其主要引起人和動(dòng)物的泌尿道感染、新生兒腦膜炎、敗血癥、肺炎、子宮內(nèi)膜炎和乳房炎。近年來,越來越多的研究顯示,動(dòng)物源ExPEC的發(fā)病率呈逐年上升趨勢,經(jīng)相關(guān)研究發(fā)現(xiàn),動(dòng)物源ExPEC與人源ExPEC毒力基因的序列相似且同源性較高[1-4],為此,對動(dòng)物源ExPEC開展比較基因組學(xué)研究,分析其動(dòng)物源ExPEC與人源ExPEC之間毒力的差異已刻不容緩。本研究對不同動(dòng)物的ExPEC進(jìn)行分離鑒定和致病性實(shí)驗(yàn),并將其中的豬源ExPEC全基因組序列與NCBI中42株大腸桿菌進(jìn)行比較基因組學(xué)分析,進(jìn)一步對其中含有的完整T6SS2型分泌系統(tǒng)中的未知功能基因vasF和vasK進(jìn)行克隆和原核表達(dá),以期對ExPEC的分子流行病學(xué)及致病機(jī)制的研究奠定基礎(chǔ)。方法與結(jié)果如下:1、采用分子生物學(xué)方法對患肺炎動(dòng)物送檢樣本中的ExPEC進(jìn)行分離鑒定,其結(jié)果顯示在5個(gè)送檢動(dòng)物的樣本中分離到ExPEC,其分別來自斑馬(BM1)、豬(ZF3,ZF7)、兔(TF)的肺臟及禽(AF1)的氣囊。采用karber法對5株Ex PEC的LD50進(jìn)行測定,其結(jié)果分別為1.28×107CFU、9.32×106CFU、9.97×106CFU、6.79×106CFU、1.20×108CFU。2、采用blastp對比分析及聚類分析將所分離的豬源ExPEC(SLPE)與42株大腸桿菌全基因組序列進(jìn)行比對,構(gòu)建其進(jìn)化樹,結(jié)果顯示,SLPE屬于B1種群,其與APEC O78親緣進(jìn)化關(guān)系最近,而與豬腸內(nèi)致病大腸桿菌UMNK88、2.3916菌株屬于不同分離群。進(jìn)一步采用mauve軟件將SLPE基因組maping到APEC O78基因組上,通過mummer軟件對比分析SLPE與APEC O78的共線性關(guān)系,結(jié)果顯示,SLPE與APEC O78的共線性良好,對其與APEC O78進(jìn)化關(guān)系較近的結(jié)果進(jìn)行了驗(yàn)證。3、采用PGAP軟件將SLPE毒力相關(guān)基因進(jìn)行聚類分析,結(jié)果顯示,雖然SLPE與APEC O78進(jìn)化關(guān)系相近,但其較APEC O78擁有更多的腸內(nèi)大腸桿菌的毒力因子。通過對毒力基因熱圖的繪制,發(fā)現(xiàn)粘附素papGⅡ、pix菌毛基因簇,鐵載體ireA和fecR,毒素/侵襲素hlyF,其他蛋白pgtP只存在于ExPEC中;SLPE還含有一個(gè)完整的T6SS 2型的Ⅵ型分泌系統(tǒng);此外,SLPE莢膜多糖合成基因簇中決定血清型的基因與大腸桿菌所有的莢膜類型都不同。4、為探究E.coli SLPE基因組中所發(fā)現(xiàn)與大腸桿菌所有的莢膜類型都不同的莢膜多糖合成基因簇(capsular polysaccharide biosynthesis gene cluster,CPS)的來源和結(jié)構(gòu)特征,將其與E.coli組1的k30和肺炎克雷伯菌的CPS進(jìn)行保守基因、非保守基因以及GC含量的對比分析,結(jié)果顯示SLPE可能是從肺炎克雷伯菌k16血清型的2069/49、UCICRE 4、MGH46或者其他k16血清型菌株的CPS水平轉(zhuǎn)移而來,其具體的致病作用以及與腸道外定植的關(guān)系還需要進(jìn)一步研究。5、通過與NCBI已公布的NMEC RS218 T6SS2型分泌系統(tǒng)進(jìn)行對比,發(fā)現(xiàn)SLPE含有一個(gè)完整的T6SS 2型分泌系統(tǒng),通過對未知功能的vasF和vasK基因進(jìn)行克隆及原核表達(dá),獲得了大小分別約為30kDa、50kDa的融合蛋白。
[Abstract]:Extraintestinal pathogenic Escherichia coli (ExPECs) is the pathogen causing serious infection in human and animal. It mainly causes urinary tract infection, neonatal meningitis, septicemia, pneumonia, endometritis and mastitis. In recent years, more and more studies have shown that the incidence of animal-derived ExPEC is on the rise year by year. It is found that the sequence of virulence gene of animal source ExPEC is similar to that of human source ExPEC with high homology [1-4]. The comparative genomics study of animal origin ExPEC and the analysis of the difference of virulence between animal origin ExPEC and human source ExPEC have been carried out without delay. In this study, ExPEC of different animals was isolated and identified and pathogenicity test was carried out, and the whole genome sequence of porcine ExPEC was compared with 42 strains of Escherichia coli in NCBI for comparative genomics analysis. Further cloning and prokaryotic expression of the unknown functional genes vasF and vasK in the complete T6SS2 secretory system were carried out in order to lay a foundation for the study of molecular epidemiology and pathogenesis of ExPEC. Methods and results were as follows: 1. ExPEC was isolated and identified by molecular biological method from the samples of animals suffering from pneumonia. The results showed that ExPECs were isolated from the lungs of zebra BM1, ZF3 ZF7, and AF1 from rabbits. The LD50 of 5 strains of Ex PEC was determined by karber method. The results were 1.28 脳 10 7 CFU 9.32 脳 10 6 CFU 9.97 脳 10 6 CFU 6.79 脳 10 6 CFU 1.20 脳 10 8 CFU 路2 respectively. The isolated porcine strain ExPECSLPE) was compared with 42 strains of Escherichia coli genome sequence by blastp comparative analysis and cluster analysis, and the phylogenetic tree was constructed. The results showed that slpe belonged to B1 population and had the closest phylogenetic relationship with Apec O78, while it belonged to different isolates from Escherichia coli UMNK882.3916. The collinear relationship between slpe and APEC O78 was analyzed by mummer software. The results showed that the collinearity of slpe and APEC O78 was good. The close relationship between SLPE and APEC O78 was verified. 3. Cluster analysis of SLPE virulence related genes was carried out by PGAP software. The results showed that the evolutionary relationship between slpe and APEC O78 was similar, the results showed that the relationship between SLPE and APEC O78 was similar to that of APEC O78. But it has more virulence factors than APEC O 78. By drawing the thermogram of virulence gene, it was found that the adhesin PAPG 鈪，

本文編號：2004018