鸭疫里默氏菌荚膜多糖输出外膜蛋白wza基因缺失株的构建及功能研究

发布时间：2018-01-25 04:58

本文关键词： 鸭疫里默氏菌基因缺失 Wza 荚膜毒力　出处：《四川农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：利用生物信息学分析软件初步确定鸭疫里默氏菌基因组中存在荚膜多糖输出基因簇及其具有多糖输出功能的wza基因,构建wza基因缺失株,初步验证wza基因在鸭疫里默氏菌在致病性、生物膜形成、抗生素抗性、抗血清杀伤作用、细胞黏附与入侵以及荚膜形成等生物学特征中所发挥的作用,主要结果如下：1 wza基因的生物信息学分析wza基因全长为726 bp,在其开放阅读框上游11 bp处具有典型TATA启动子结合位点。此外,在wza基因下游,具有调节荚膜多糖输出的酪蛋白激酶wzc基因、多个荚膜多糖合成相关的糖基转移酶基因以及脂质合成相关基因。该基因所编码的Wza蛋白由241个氨基酸组成、分子量约26.6KD。经过氨基酸序列比对分析,发现Wza蛋白与黄杆菌科细菌的多糖转运子具有较高相似性。保守功能域搜索发现,该蛋白具有Poly_export family功能域,其主要功能是负责多糖的输出。亚细胞定位预测发现其为一个外膜蛋白中的荚膜多糖输出外膜蛋白Wza。Wza蛋白的二级结构主要组成成分为：α螺旋(23.24%)、延伸链(30.29%)、β折叠(14.11%)以及无规则卷曲(32.37%)。最后,使用同源建模的方法,使用SWTSS-MODEL程序构律了鸭疫早默氏菌Wza蛋白的三级结构。2鸭疫里默氏菌CH-1Δwza缺失株的构建本次缺失株的构建：利用自杀质粒pYA4278通过同源重组的方法将wza基因的开放阅读框中的704 bp敲掉。采用重叠PCR技术构建wza基因左臂、右臂、spc三片段的融合PCR片段Awza-spc。自杀质粒pYA4278经AdhI酶切后具有T克隆载体的T粘性末端特征,本文直接将Awza-spc融合片段克隆到pYA4278上构建了pYA4278-Δwza-spc重组自杀质粒。将含有pYA4278-Δwza-spc的大肠杆菌X7123与鸭疫里默氏菌CH-1株进行接合转移,筛选获得CH-1Δwza缺失株并经PCR鉴定正确。3鸭疫里默氏菌wza基因的部分功能初探wza基因的缺失导致鸭疫里默氏菌的生长减慢,其菌落特征基本无变化。使用透射电镜观察发现,缺失株的荚膜明显变薄。结晶紫染色分析发现鸭疫里默氏菌CH-1是一株典型的生物膜缺陷株,其OD595仅为0.049；而CH-1 Δwza的生物膜形成能力却明显的增加,其OD595为0.269。荚膜主要成分是荚膜多糖,其具有很强的亲水性效果和抗氧化能力。通过抗干燥试验以及抗氧化试验的结果可以看出,缺少荚膜保护的CH-1 Δwza在干燥环境或者低浓度的双氧水条件下的存活率远低于亲本株。此外,表面疏水性试验结果显示缺失株的疏水性显著升高。选择常用的抗生素药敏纸片检测CH-1 Δwza和其亲本株的对抗生素的敏感性的差异,与亲本株相比,CH-1 Δwza对大部分抗生素的敏感性有明显提高,表明鸭疫里默氏菌荚膜多糖的荚膜与抗生素的抗性存在联系：其荚膜与多种细菌一样,是抵抗抗生素的天然保护屏障。补体杀伤试验表明亲本株在90%血清浓度下的存活率大约为85%,而缺失株的存活率为0。说明wza基因在鸭疫里默氏菌侵染宿主以及引起败血症方面发挥着巨大作用。4.Wza是鸭疫里默氏菌的一个毒力因子细菌对宿主细胞的黏附和入侵是细菌感染宿主的先决条件,而wza基因的缺失致使鸭疫里默氏菌细胞黏附力下降,数据显示CH-1 Δwza和亲本株在黏附试验中的黏附菌落数分别为5.8×104 CFU/孔和3.6x104CFU/孔。此外,通过测定CH-1 Δwza与亲本株的LD50,发现亲本株的LD50为5.62×107CFU/mL,而缺失株CH-1△wza的LD50为2.37x1010 CFU/mL。根据LD50值可以得出wza基因的缺失导致了鸭疫里默氏菌毒力下降了421倍。因此,wza基因与鸭疫里默氏菌的致病性具有一定的联系。
[Abstract]:Using bioinformatics analysis identified Riemerella anatipestifer genomes in presence of capsular polysaccharide export gene cluster and Wza gene has a polysaccharide output function, the construction of Wza gene deletion strains, preliminary verification of Wza gene in Riemerella anatipestifer in pathogenicity, biofilm formation, antibiotic resistance, antiserum killing effect, play cell the adhesion and invasion as well as the capsule forming the biological characteristics of the role, the main results are as follows: 1 Wza gene bioinformatics analysis of Wza gene was 726 BP in the open reading frame of 11 bp upstream of typical TATA promoter binding sites. In addition, in the downstream of the Wza gene, with casein kinase WZC gene regulates capsular polysaccharide sugar output, a plurality of capsular polysaccharide synthesis related glycosyltransferase gene and lipid synthesis genes. The gene encoding the Wza protein consists of 241 amino acids, molecular The amount of about 26.6KD. by amino acid sequence analysis found that the polysaccharide transporter protein Wza and flavobacteriaceae bacteria have high similarity. Conserved domain search found that the protein has a Poly_export domain of family, its main function is to output for polysaccharide. Subcellular localization prediction showed that the two level structure of a membrane protein the output of the capsular polysaccharide outer membrane protein Wza.Wza protein as main component: the alpha helix (23.24%), extended chain (30.29%), (14.11%) and beta folding random coil (32.37%). Finally, using homology modeling method, using the SWTSS-MODEL program. The law construction of duck plague early Alzheimer's strain Wza protein the three level structure of.2 Riemerella anatipestifer CH-1 Delta Wza mutant to construct the mutant by homologous recombination with the Wza gene open reading frame of 704 BP knocked out by plasmid pYA4278. The Dutch act Construction of Wza gene PCR technology stack left arm, right arm, T sticky ends feature fusion fragment of PCR Awza-spc. SPC three Dutch act fragments of plasmid pYA4278 were digested with AdhI with T cloning vector, this paper will directly to pYA4278 to construct pYA4278- recombinant plasmid wza-spc Dutch act fusion Awza-spc fragment was cloned. PYA4278- containing a wza-spc of Escherichia coli X7123 with Riemerella anatipestifer strain CH-1 conjugation, missing part function of Wza gene CH-1 Delta Wza mutant was screened and identified by PCR.3 of Riemerella anatipestifer Wza gene leads to decreased growth of Riemerella anatipestifer, no change in the basic characteristics of colonies. TEM observation showed that the capsule deficient strains was thinner. Crystal violet staining analysis found that Riemerella anatipestifer CH-1 is a typical biofilm mutant, the OD595 is only 0.049; and the biofilm forming ability was CH-1 Wza Increased significantly, the OD595 as the main component of 0.269. capsule is capsular polysaccharide, which has hydrophilic effect and strong antioxidative capability. The anti drying test and antioxidant test results show that the survival rate of the lack of protection of the CH-1 Wza capsule in the double oxygen water dry environment or the low concentration of the far below the parental strain. In addition, the surface hydrophobicity test showed that hydrophobic deletion strains were significantly increased. Differences in antibiotic susceptibility test Wza CH-1 a commonly used and its parental strains of antibiotic sensitivity, compared with the parent strain, CH-1 Wza has significantly improved sensitivity to most antibiotics, indicate the presence of contact resistance the capsule and antibiotics of Riemerella anatipestifer capsular polysaccharide: the capsule with a variety of bacteria, is a natural protective barrier against antibiotics. The complement test showed that parental strain in the killing of 90% serum The concentration of the survival rate is about 85%, while the mutant survival rate was 0. in the Wza gene of Riemerella anatipestifer infection and septicemia play a huge role in.4.Wza is a bacterial virulence factor of Riemerella anatipestifer on host cell adhesion and invasion is a prerequisite for host bacterial infection. The deletion of Wza gene in Riemerella anatipestifer decreased cell adhesion, adhesion data show colonies strain in adhesion test in the CH-1 Delta Wza and 5.8 * 104 CFU/ lines and 3.6x104CFU/ holes respectively. In addition, through the determination of CH-1 Wza and parental strain LD50, found that parental strain LD50 was 5.62 * 107CFU/mL however, the mutant strain CH-1 LD50 2.37x1010 CFU/mL. Wza LD50 according to the missing values can be obtained in the Wza gene of Riemerella anatipestifer virulence decreased 421 times. Therefore, the pathogenicity and Wza gene of Riemerella anatipestifer has A certain connection.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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