空肠弯曲菌galE及Cj1136、Cj1138、Cj1139基因序列对比研究

发布时间：2018-04-26 09:07

本文选题：格林—巴利综合征 + 空肠弯曲菌　；参考：《河北医科大学》2010年硕士论文

【摘要】： 目的:空肠弯曲菌(Campylobacter jejuni, C. jejuni)是世界范围内导致细菌性腹泻的主要病原菌之一,部分格林—巴利综合征(Guillain-Barre syndrome, GBS)是发生于C. jejuni感染后的周围神经疾病。根据不同的神经生理学和病理学特点,GBS被分为急性炎症性脱髓鞘性多发性神经病(AIDP)、急性运动轴索性神经病(AMAN)、和Miller-Fisher综合征(MFS)等几种亚型。自2000年C.jejuni NCTC11168菌株的全基因序列完成以来,现已有5株C.jejuni的全基因组序列发表在GeneBank中。现有大量研究表明空弯表面的脂寡糖(LOS)成分和周围神经的神经节苷脂之间的分子模拟导致免疫损伤是空弯感染后GBS的主要发病机制。galE及Cj1136、Cj1138、Cj1139基因编码的多种蛋白参与了细菌表面LOS的生物合成。本研究从基因突变的角度出发,选择华北地区具有致GBS特性的三株空肠弯曲菌菌株,对其LOS合成相关的galE及Cj1136、Cj1138、Cj1139基因序列进行测定,以了解华北地区致轴索型GBS的空弯菌株核酸序列特征,并同GenBank中的空弯菌株相应序列进行对比分析,寻找基因突变和变异与致病性的关系,及菌株的基因地域特点并分析其遗传进化关系。方法:细菌培养选取分离自华北地区AMAN型GBS病人的C.jejuni菌株。血清分型为pennerO:19型,pennerO:2型,pennerO:5型。取C.jejuni专用CM0739培养基(Oxid公司英国),按说明书制备固体培养基。将菌株接种于CM0739固体培养基,置于厌氧培养箱中,在37℃微需氧条件下培养,传代48-72 h收菌。基因DNA组提取应用promega公司的Wizard基因提取试剂盒,按照试剂盒说明提取C.jejuni基因组DNA。序列测定将细菌基因组DNA送至Analytical Genetics Technology Centre, University Health Network,Toronto Princess Margaret Hospital及深圳华大基因研究院测序。序列对比和进化分析以NCTC11168为对照寻找存在的突变并应用Mega version 4软件,将3株C.jejuni的galE、Cj1136、Cj1138、Cj1139基因序列同BLAST获得序列的的相应位点及进行多序列对比,寻找变异碱基,计算序列间的遗传距离并以Neighbor -Joining(NJ)法构建进化树。结果:三株C. jejuni菌株的galE基因由987个碱基构成;Cj1136基因由1173个碱基构成;Cj1138基因由1170个碱基构成;Cj1139基因由912个碱基构成,与NCTC11168的galE及Cj1136、Cj1138、Cj1139基因序列相比,此3株致GBS C. jejuni中galE基因核苷酸序列有4个相同碱基突变并导致了4个对应的相同氨基酸突变。遗传距离计算,qiaoyuntao株、zhanxing株及lulei株的galE基因序列分别与CF93-6、CF93-6及HB93-13株的相似程度最高,遗传距离分别为0.1%、0.2%,0.6%。zhanxing株与qiaoyuntao株距离为1.5%,zhanxing株与lulei株距离为1.6%,qiaoyuntao株与lulei株距离为0.5%。此3株致GBS C. jejuni中Cj1138存在2个相同的碱基突变并导致了2个对应的相同氨基酸突变。遗传距离计算,3株致GBS C. jejuni的Cj1138基因之间最大遗传距离为2.1%。结论:与NCTC11168相比GBS相关C. jejuni中galE及Cj1138基因序列存在相同碱基突变,这些碱基突变可能与C. jejuni致GBS能力的改变相关。该3株致GBS C. jejuni的galE、Cj1136、Cj1138、Cj1139基因遗传距离较小,反映河北地区致GBS的C. jejuni在进化上存在聚类现象,具有一定的区域特征。
[Abstract]:Objective: Campylobacter jejuni (C. jejuni) is one of the major pathogenic bacteria causing bacterial diarrhea worldwide, and partial Green Barre syndrome (Guillain-Barre syndrome, GBS) is a peripheral nerve disease occurring after C. jejuni infection. According to different neurophysiological and pathological characteristics, GBS is divided into acute disease. Inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), and Miller-Fisher syndrome (MFS), and other subtypes of Miller-Fisher syndrome (MFS). Since the completion of the whole gene sequence of C.jejuni NCTC11168 in 2000, a total of 5 C.jejuni genome sequences have been published in GeneBank. Molecular simulation between oligosaccharide (LOS) components and gangliosides of peripheral nerve leads to immune damage as the main pathogenesis of GBS after empty bending infection,.GalE and Cj1136, and a variety of proteins encoded by Cj1138 and Cj1139 genes participate in the biosynthesis of the bacterial surface LOS.
In this study, three strains of Campylobacter jejuni with GBS characteristics in North China were selected from the angle of gene mutation to determine the sequence of galE, Cj1136, Cj1138 and Cj1139 genes related to its LOS synthesis, in order to understand the sequence characteristics of the nucleic acid of the axonal GBS in North China, and to sequence with the empty bending strain in GenBank. Comparative analysis was conducted to find out the relationship between gene mutation and mutation and pathogenicity, as well as the regional characteristics of the strains and analyze their genetic evolution relationship.
Methods: bacterial culture was used to select C.jejuni strains isolated from AMAN type GBS patients in North China. The serotypes were classified into pennerO:19, pennerO:2, pennerO:5 type, and C.jejuni special CM0739 medium (Oxid UK). The solid medium was prepared according to the instructions. The strains were inoculated to CM0739 solid medium and placed in the anaerobic incubator, at 37 degrees centigrade. Under the oxygen condition, the bacteria were collected at 48-72 h.
Gene DNA group was extracted from Promega gene Wizard extraction kit, and C.jejuni genomic DNA. was extracted according to the kit instructions.
Sequencing of bacterial genome DNA was sent to Analytical Genetics Technology Centre, University Health Network, Toronto Princess Margaret Hospital and the Shenzhen Huada gene Institute for sequencing.
Sequence contrast and evolutionary analysis search for existing mutations with NCTC11168 as control and use Mega version 4 software to compare the sequences of galE, Cj1136, Cj1138, Cj1139 gene of 3 strains of C.jejuni with BLAST sequence and perform multiple sequence comparison, find variant bases, calculate the genetic distance between sequences, and construct a Neighbor -Joining (NJ) method. Build a tree of evolution.
Results: the galE gene of three strains of C. jejuni was composed of 987 bases, and the Cj1136 gene was composed of 1173 bases, and the Cj1138 gene was composed of 1170 bases, and the Cj1139 gene was composed of 912 bases. The nucleotide sequences of the 3 strains of galE and Cj1136, Cj1138 and Cj1139 were 4 of the same base sequences. The mutation resulted in 4 corresponding amino acid mutations. The genetic distance was calculated. The galE gene sequences of qiaoyuntao, Zhanxing and Lulei strains were similar to CF93-6, CF93-6 and HB93-13, respectively, with the genetic distance of 0.1%, 0.2%, 0.6%.zhanxing and qiaoyuntao 1.5%, Zhanxing and Lulei 1.6%, Qi, and Qi. The distance between the aoyuntao strain and the Lulei strain was 0.5%. of the 3 strains of GBS C. jejuni and Cj1138 existed 2 same base mutations and resulted in 2 corresponding amino acid mutations. The genetic distance was calculated, and the maximum genetic distance between the 3 GBS C. jejuni Cj1138 genes was 2.1%..
Conclusion: there is the same base mutation in the galE and Cj1138 gene sequences in GBS related C. jejuni compared with NCTC11168. These base mutations may be related to the change of GBS ability caused by C. jejuni. The 3 strains of GBS C. jejuni have smaller genetic distance. The image has certain regional characteristics.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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