鼠疫耶尔森菌编码序列及田鼠型鼠疫菌致病相关基因研究

发布时间：2018-06-07 18:45

本文选题：鼠疫耶尔森菌 + 编码序列　；参考：《中国疾病预防控制中心》2009年硕士论文

【摘要】： 鼠疫是一种自然疫源性疾病,曾引起三次世界性的大流行,我国发生过6次较大规模的鼠疫流行,发病人数超过百万。目前,我国存在着12种类型的鼠疫疫源地,鼠疫菌可分为18个生态型。其中,布氏田鼠和青海田鼠鼠疫菌具有区别于其它类型鼠疫菌的特点,对小白鼠等小型动物毒力强,对豚鼠、兔、羊等大型动物几乎无毒,从发现至今,尚无关于该类鼠疫菌感染人类的相关报道。而对于田鼠型鼠疫菌的特殊致病力,一直是亟待解决的问题。 2005年,云南省玉龙县发生了一起肺鼠疫疫情,发病5人,死亡2人。在随后的鼠疫流行病学调查过程中,在当地的啮齿类动物中分离到了鼠疫菌,确定了玉龙为新发鼠疫自然疫源地。为了进一步了解这一新发疫源地性质和流行规律,对所分离鼠疫菌及相邻疫源地鼠疫菌进行了全基因组序列测定和分析。本研究通过对全世界不同来源鼠疫菌的全基因组序列分析,获得了已测序各株鼠疫菌编码序列(CDS)的两两比较结果,并确定了CDS之间一一对应的同源关系。在此基础上,通过对玉龙及相邻疫源地鼠疫菌间的CDS、SNP和基因组重排的比较,显示玉龙菌株D106004与西藏菌株Z176003 CDS的同源性最高,重排片段最少,亲缘关系最相近。对于所测序三株菌在生化性状上的差异,利用全基因组序列资料,对麦芽糖相关基因进行了搜索比较,并根据突变位点情况在我国72株鼠疫菌中进行实验研究,发现malT、malZ两个基因在我国部分麦芽糖酵解阴性的鼠疫菌株中存在基因的移码突变,推测这两个基因的突变可能是引起我国部分鼠疫菌株麦芽糖酵解阴性的原因。根据所获得的CDS分析比较结果,根据致病基因在强毒株中表现为一种类型,在弱毒株中则表现另外一种类型的特点,寻找田鼠型菌株特殊致病力相关突变基因,发现91001(布氏田鼠鼠疫菌测序菌株)特殊致病力相关突变基因21个。对21个基因突变位点设计引物,在我国两类田鼠型鼠疫菌和其它疫源地鼠疫菌共131株进行研究验证,实际比较这些基因的分布和变化情况。实验结果显示,1)3个基因(T2,T18,T24基因均为点突变)事实上在我国的两类田鼠型鼠疫菌中均未发生突变。2)T13基因点突变,在部分布氏田鼠鼠疫菌中未发生突变。3)2个基因(T30点突变,Q5基因缺失)对照组鼠疫菌株中基因表现不一致。4)3个基因(S1、T29基因点突变,T3基因缺失)在布氏田鼠鼠疫菌中发生了突变,而青海田鼠鼠疫菌未发生变化,这一特征具有分型和溯源的流行病学意义。5)确证12个基因(9个点突变,3个基因缺失)在所有田鼠型鼠疫菌中均发生了突变,这12个基因可能是引起田鼠型菌株特殊致病力的突变基因,其中有2个基因突变表现为基因缺失,功能与信息储存加工相关,属于变异较快的基因类型,一个T6基因功能注释为DEAD盒蛋白家族的解螺旋酶,另一个T8基因注释为AraC蛋白家族的转录调控蛋白,它们均与细菌的致病力相关,在下一步的研究中可以对这些基因进行功能研究。 T6基因在我国田鼠型鼠疫菌中较其它疫源地的鼠疫菌缺失了510bp的片段而发生了较大的突变,其可能对田鼠型鼠疫菌在血清中的生长速度产生影响。在进一步的研究中,采用基因一步缺失法对T6基因进行缺失替换,以判断其对田鼠型鼠疫菌的致病性所产生的实际影响。
[Abstract]:Plague, a natural epidemic disease, has caused three worldwide pandemics, and there have been 6 large scale plague epidemics in China. There are 12 types of plague foci in our country, and Yersinia pestis can be divided into 18 ecotypes. Among them, the plague bacteria of Bryonia bryoni and Qinghai vole are different from other types The characteristics of Yersinia pestis are very toxic to small mice and other small animals, and they are almost non-toxic to large animals such as guinea pigs, rabbits and sheep. Since the discovery, there has been no related report on the infection of the Yersinia pestis. The special pathogenicity of Yersinia pestis has been a problem to be solved urgently.
In 2005, a lung plague epidemic occurred in ERON County, Yunnan Province, with 5 people and 2 deaths. In the subsequent epidemiological investigation of the plague, Yersinia pestis was isolated in the local rodent, and ERON was identified as the natural foci of the new plague. Complete genome sequencing and analysis of Yersinia pestis and its adjacent foci were carried out.
In this study, by analyzing the whole genome sequence of Yersinia pestis from different sources of the world, the 22 comparison results of the sequence of CDS were obtained, and the corresponding homologous relationship between CDS was determined. On this basis, the comparison of CDS, SNP and genome rearrangement between ERON and the adjacent Phytophthora was compared. The results showed that ERON strain D106004 had the highest homology with Tibet strain Z176003 CDS, and the rearrangement fragment was the least.
For the differences in the biochemical characters of the three strains, the whole genome sequence data were used to search for the maltose related genes, and the 72 strains of Yersinia pestis were studied in China according to the mutation site. It was found that the genes of malT, malZ and two genes in the plague strains of maltose glycolysis negative in our country Frameshift mutation suggests that the mutation of these two genes may be the cause of maltoglycolysis of some plague strains in China.
According to the results obtained by CDS analysis, according to the manifestation of the pathogenic gene in the strong virulent strain, the special pathogenicity related mutation gene of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the 91001 strains of the strain of the brucellus plague sequence were found to be 21. A total of 131 strains of Yersinia pestis and other Phytophthora Phytophthora of two kinds of voles in China were studied and verified by the mutation site design primers, and the distribution and change of these genes were compared.
The experimental results showed that, 1) 3 genes (T2, T18, T24 genes were point mutations) in fact, there were no mutation.2 in the two types of Yersinia pestis in China, the mutation of the T13 gene, the 2 genes (T30 point mutation, Q5 based deficiency) in the pestis strain of a part of the brucellus Yersinia pestis, 3 genes in the Yersinia pestis strain S1, T29 gene point mutation, T3 gene deletion) occurred in the Brandt vole Yersinia pestis, and the Qinghai vole Yersinia pestis did not change, this characteristic has the typing and traceability epidemiological significance.5) confirmed that 12 genes (9 point mutation, 3 gene deletion) have been mutated in all field Yersinia pestis, the 12 genes may be It is a mutant gene that causes the special virulence of the strain of the strain of the vole. 2 of the mutations are gene deletion, and the function is related to the processing of information storage. It belongs to the fast variant gene type. One T6 gene function is annotated by the DEAD box protein family of helicase and the other T8 gene is the transcriptional regulation protein of the AraC protein family. They are all related to the pathogenicity of bacteria, which can be studied in the next step.
The T6 gene in the Yersinia pestis of our country is less than the 510bp fragment in the other foci of Yersinia pestis, which may affect the growth speed of the Yersinia pestis in the serum. In the further study, the one step deletion method of gene is used to replace the T6 gene, so as to judge the rat model of the voles. The actual effects of the pathogenicity of Phytophthora.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378

【引证文献】