猪致肺炎大肠杆菌比较基因组学分析和T6SS分泌系统vasF、vasK的表达

发布时间：2018-06-10 17:26

  本文选题：肠外致病性大肠杆菌大肠杆菌 + 比较基因组　； 参考：《吉林农业大学》2015年硕士论文

【摘要】：肠外致病性大肠杆菌(Extraintestinal pathogenic Escherichia coli,ExPEC)是引起人和动物肠道外严重感染的病原体,其主要引起人和动物的泌尿道感染、新生儿脑膜炎、败血症、肺炎、子宫内膜炎和乳房炎。近年来,越来越多的研究显示,动物源ExPEC的发病率呈逐年上升趋势,经相关研究发现,动物源ExPEC与人源ExPEC毒力基因的序列相似且同源性较高[1-4],为此,对动物源ExPEC开展比较基因组学研究,分析其动物源ExPEC与人源ExPEC之间毒力的差异已刻不容缓。本研究对不同动物的ExPEC进行分离鉴定和致病性实验,并将其中的猪源ExPEC全基因组序列与NCBI中42株大肠杆菌进行比较基因组学分析,进一步对其中含有的完整T6SS2型分泌系统中的未知功能基因vasF和vasK进行克隆和原核表达,以期对ExPEC的分子流行病学及致病机制的研究奠定基础。方法与结果如下:1、采用分子生物学方法对患肺炎动物送检样本中的ExPEC进行分离鉴定,其结果显示在5个送检动物的样本中分离到ExPEC,其分别来自斑马(BM1)、猪(ZF3,ZF7)、兔(TF)的肺脏及禽(AF1)的气囊。采用karber法对5株Ex PEC的LD50进行测定,其结果分别为1.28×107CFU、9.32×106CFU、9.97×106CFU、6.79×106CFU、1.20×108CFU。2、采用blastp对比分析及聚类分析将所分离的猪源ExPEC(SLPE)与42株大肠杆菌全基因组序列进行比对,构建其进化树,结果显示,SLPE属于B1种群,其与APEC O78亲缘进化关系最近,而与猪肠内致病大肠杆菌UMNK88、2.3916菌株属于不同分离群。进一步采用mauve软件将SLPE基因组maping到APEC O78基因组上,通过mummer软件对比分析SLPE与APEC O78的共线性关系,结果显示,SLPE与APEC O78的共线性良好,对其与APEC O78进化关系较近的结果进行了验证。3、采用PGAP软件将SLPE毒力相关基因进行聚类分析,结果显示,虽然SLPE与APEC O78进化关系相近,但其较APEC O78拥有更多的肠内大肠杆菌的毒力因子。通过对毒力基因热图的绘制,发现粘附素papGⅡ、pix菌毛基因簇,铁载体ireA和fecR,毒素/侵袭素hlyF,其他蛋白pgtP只存在于ExPEC中;SLPE还含有一个完整的T6SS 2型的Ⅵ型分泌系统;此外,SLPE荚膜多糖合成基因簇中决定血清型的基因与大肠杆菌所有的荚膜类型都不同。4、为探究E.coli SLPE基因组中所发现与大肠杆菌所有的荚膜类型都不同的荚膜多糖合成基因簇(capsular polysaccharide biosynthesis gene cluster,CPS)的来源和结构特征,将其与E.coli组1的k30和肺炎克雷伯菌的CPS进行保守基因、非保守基因以及GC含量的对比分析,结果显示SLPE可能是从肺炎克雷伯菌k16血清型的2069/49、UCICRE 4、MGH46或者其他k16血清型菌株的CPS水平转移而来,其具体的致病作用以及与肠道外定植的关系还需要进一步研究。5、通过与NCBI已公布的NMEC RS218 T6SS2型分泌系统进行对比,发现SLPE含有一个完整的T6SS 2型分泌系统,通过对未知功能的vasF和vasK基因进行克隆及原核表达,获得了大小分别约为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，

本文编号：2004018