布鲁氏菌中国分离株遗传多态性研究

发布时间：2018-02-04 03:57

本文关键词： 布鲁氏菌基因分型 DFR MLST MLVA　出处：《吉林大学》2012年博士论文　论文类型：学位论文

【摘要】：布鲁氏菌病是由布鲁氏菌侵入机体引起的一类传染－变态反应性的全球性分布的人畜共患病，在许多发展中国家流行并造成很大的经济损失。依据对宿主偏好性以及培养和生物学特性的不同，布鲁氏菌分为6个经典的种，其中羊种、牛种、猪种、犬种4个种能够使人致病。布鲁氏菌基因的高度保守性使得建立种型之间的遗传关系极具挑战性。了解布鲁氏菌基因水平的差异，有助于了解布鲁氏菌的进化与毒力特征。目前布鲁氏菌基因分型方法中以多位点序列分型(MLST)和多位点VNTR (MLVA)法重复性好、分辨率高，但是由于布鲁氏菌的变异，在实际工作中已遇到问题。寻找一种更好的分型方法或者是多种方法的联合运用，可能会更好地解决布鲁氏菌的分种分型问题。通过十株已测序的布鲁氏菌全基因组的比较分析和布鲁氏菌全基因组芯片杂交的结果，共鉴定出53个差异基因区段（DFR），其中位于I号染色体上的DFR有26个，，II号染色体上的DFR为27个，II号染色体的DFR密度远远高于I号染色体。探讨这些差异区段在19株标准菌株和129株我国不同种型布鲁氏菌分离株的分布特征表明，差异区段可能是进化过程中获得或缺失的，与菌株的遗传关系没有必然的联系，为研究不同疫源地或不同年代的布鲁氏菌菌株基因水平差异提供了依据。为了提高传统的MLST方法（CMLST）的分型分辨率，我们在本研究中探讨通过加长基因的PCR扩增片段来用于MLST分型研究，即为改良的MLST方法（EMLST）。对129株我国布鲁氏菌分离株进行了CMST和EMLST的分析，用CMLST方法定义了23个ST型，而用EMLST方法则定义了35个ST型，多种方法分析表明，EMLST较CMLST具有更高的分型分辨率。和已报道的国外布鲁氏菌菌株的CMLST结果比较分析发现，我国布鲁氏菌分离株具有自己的基因型构成特点。基于看家基因的改良的MLST方法适合于亲缘关系较远的布鲁氏菌菌株的分型。传统的MLVA方法采用PCR扩增并电泳区分的方法，但是存在不稳定及费时费力的缺点，因此本研究采用测序的方法对120株分离株的15个多态性较高的MLVA位点进行分析，共分为87个型。基于VNTR位点的改良的MLVA方法应用于我国布鲁氏菌分离株的结果表明，该方法适合于亲缘关系较近的布鲁氏菌菌株的分型。对120株CMLST、EMLST以及MLVA数据综合比较分析表明，EMLST较CMLST具有更多的分支，有着更高的分辨率，而MLVA则是三种分型方法中分辨率最高的方法。
[Abstract]:Brucellosis is a class of infectious-allergic global zoonosis caused by brucellosis. Brucellosis is prevalent in many developing countries and causes great economic losses. According to the difference of host preference, culture and biological characteristics, Brucella is divided into six classical species, sheep, cattle and pig. The highly conserved nature of Brucella genes makes it challenging to establish genetic relationships among species. Understand the differences in Brucella gene levels. It is helpful to understand the evolutional and virulence characteristics of Brucella. At present, the methods of multilocus sequence typing (MLSTs) and multilocus VNTR (MLVA) methods have good reproducibility. High resolution, but because of the variation of brucella, has encountered problems in practical work. To find a better method of typing or combination of multiple methods. It may be better to solve the classification problem of Brucella. Based on the comparative analysis of the whole genome of ten strains of Brucella and the results of whole genome chip hybridization of Brucella, a total of 53 differentially expressed gene regions (DFR) were identified. Among them, there are 26 DFR on chromosome I and 27 DFR on chromosome II. The DFR density of chromosome II was much higher than that of chromosome I. the distribution characteristics of these differential regions in 19 standard strains and 129 strains of Brucella strains of different species in China were studied. The differential region may be acquired or missing in the course of evolution, which is not necessarily related to the genetic relationship of the strains, which provides a basis for the study of gene level differences of Brucella strains in different foci or in different years. In order to improve the typing resolution of the traditional MLST method, we studied the use of the extended PCR fragment in the study of MLST typing. The CMST and EMLST of 129 strains of Brucella in China were analyzed by CMST and EMLST for the improved MLST method. 23 St types were defined by CMLST method. In contrast, 35 St types were defined by EMLST method. EMLST has higher typing resolution than CMLST. The results of CMLST analysis of Brucella strains have been compared and analyzed. Brucella isolates in China have their own genotypic characteristics. The improved MLST method based on housekeeping gene is suitable for the genotyping of Brucella strains with distant genetic relationship. The traditional MLVA method is based on PCR amplification and electrophoresis, but it is unstable and time-consuming. Therefore, 15 polymorphic MLVA loci of 120 isolates were analyzed by sequencing. The modified MLVA method based on VNTR locus was applied to Brucella isolates in China. This method is suitable for the genotyping of brucella strains with close genetic relationship. The comprehensive analysis of 120 strains of CML-ST-EMLST and MLVA data shows that this method is suitable for the typing of Brucella strains. EMLST has more branches and higher resolution than CMLST, and MLVA is the highest resolution of the three typing methods.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378

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