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幽门螺杆菌基因敲除方法的建立及其应用研究

The Establishment of Helicobacter Pylori Gene Knockout and Application

作者: 专业:临床兽医学 导师:李家奎 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

H.pylori, cagA, hp0596, ureB, Gene knockout

        目的:幽门螺杆菌(Helicobacter pylori, H. pylori)是一种革兰氏阴性的微需氧菌,他定植于胃黏膜上皮细胞的表面,以及胃小凹内黏液深层。大量流行病学的研究调查显示:全世界约有一半的人口中存在H. pylori的感染,它与慢性胃炎、消化性溃疡、胃黏膜相关淋巴样组织淋巴瘤以及胃腺癌等疾病的发生有关联。大量研究也证实,多个H.pylori的毒力因子直接或间接地参与了上述的病理反应,大部分主要集中于研究CagA, VacA,UreB等毒力因子的研究方面,并且在H. pylori致病过程中也可能还存在其他重要的毒力因子。本研究旨在通过建立H. pylori体内基因敲除方法来筛查新的H. pylori致胃病相关蛋白,并对其进行基因改造和分析评价,以及对其致病机制进行进一步研究。方法与结果:第一部分,我们选择cagA,hp0596作为候选基因,从H. pylori 26695基因组中分别扩增得到此研究所需基因的上下游同源臂,与两端带有FRT位点的氯霉素抗性基因片段共同构建突变载体;以突变载体为模板扩增打靶片段,将其转化入H.pylori26695,在抗生素选择压力下,打靶片段和H. pylori菌体基因组发生同源重组,通过氯霉素抗性筛选得到带有抗性标记的重组菌转化子,并通过一系列的实验建立了在幽门螺杆菌中进行基因打靶的技术方法。确定了实验研究过程中的几个关键点,如打靶质粒的浓度、高效的电转化条件、所用抗生素的浓度和突变株的筛选培养方式,建立了相对高效的基因打靶的方法。在此基础上我们成功地对cagA, hp0596基因进行了敲除,并在基因组水平、RNA水平和蛋白质水平进行了验证。第二部分,为避免H. pylori重组菌株对抗生素选择压力的依赖,我们利用反选择标记sacB基因在幽门螺杆菌中建立了无痕敲除系统,即通过体内基因敲除的方式构建了打靶载体,并对其生长表型作了综合评价分析。具体而言,我们选定ureB基因作为我们进行基因敲除的目标,利用同源重组原理,成功敲除了幽门螺杆菌中ureB基因,并且结合反选择系统sacB基因的作用,得到不含抗性标记的突变株H.pylori26695(AureB)。研究表明与野生菌株相比突变菌株尿素酶的功能缺失,这样通过正负两步置换选择法在幽门螺杆菌中建立了体内基因无痕敲除的方法。第三部分,构建穿梭载体,实现Salmonella的肠黏附素蛋白在HPSS1株外膜蛋白上的表达。利用λRed系统将穿梭质粒pHH43进行改造,将菌蜕基因genE及其上游的温度敏感型启动子和下游的cat抗性基因,通过同源重组的方法置换为Salmonella的肠黏附素操纵元1pfABCDE,并将筛选基因替换为卡那霉素抗性基因。将改造好的质粒转化入HPSSl株中,实现黏附素1pfA蛋白在外膜的表达,并开展后续的鉴定工作。结论:在幽门螺杆菌中建立了高效的体内基因重组方法,证明此方法对研究分析幽门螺杆菌的功能是最有力的工具。并且利用λRed系统成功构建改造了E. coli-H.phlori穿梭表达载体,实现了lpfA蛋白在外膜的表达。
    Objective:Helicobacter pylori is a micro-aerophilic, Gram-negative bacterium that can efficiently colonizes the gastric epithelial cells or gastric mucin. It is estimated that H.pylori infects at least half of the world’s population. The chronic, persistent infection by H.pylori may cause many gastric diseases including chronic atrophic gastritis, peptic ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. H.pylori is equipped with numerous virulence factors, which facilitate the colonization and damage of gastric mucosa. Some of the virulence factors have been identified such as CagA, VacA and UreB, while many other factors that maybe important in the pathogenesis are still unknown. The aim of this study was to identify novel gastric disease-related antigens using in vivo knock-out approach from H.pylori and characterize their role in the pathogenesis as well.Methods and result:In section one, cagA and hp0596 were selected as the target genes for in vivo gene knock-out in H.pylori. Upstream and downstream homologous fragments of the target genes from H.pylori26695 as well as chloramphenicol resistance cassette flanked with two FRT sites were amplified, then all the fragments were inserted into plasmid pBlueskript skⅡ(+) to construct a recombinant vector. The target cassette then amplified from the recombinant vector and transformed into H.pylori26695. Under the pressure of antibiotic selection, homologous recombination occurred between the cassette and H.pylori chromosome, and the final recombinants were selected on chloramphenicol agar plate. The mutant strains were verified by colony PCR. Some basic factors were optimized, such as temperature-sensitive replication vector, length of homologous regions, high-efficiency electroporation protocol, antibiotic concentration, and the method for identification of positive recombinant. By using this procedure, two candidate genes cagA and hp0596 were knocked out from the chromosome. The mutant strains were verified by colony PCR, RT-PCR, total protein SDS-PAGE and western blot. All the results demonstrated that cagA and hp0596 gene was successfully deleted from H.pylori chromosome.In section two, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows the introduction of unmarked mutations into H.pylori. A comprehensive phenotypic analysis of the mutants was explored subsequently. Specifically, we obtained urease protect and assist its colonization played an important role in regard to a key gene responsible for in H.pylori through bioinformatic analysis. According to homologous recombination, ureB was deleted successfully. Combined with counterselection system the markerless mutant was gained, which named H.pylori26695△ureB and urease enzyme activity had been lost. This result raised the possibility that we could exploit the sucrose sensitivity phenotype conferred by sacB expression to perform a functional exchange of ureB alleles.In the third part of this work, we construct a E. coli-H. pylori shuttle vector in order to achieve expression of heterogenous intestinal adhesion protein on outer membrane of HPSS1. We use theλRed recombination system to reform the shuttle plasmid pHH43, in brief, the bacterial ghosts gene GenE together with the temperature-sensitive promoter and the chloramphenicol resistance gene of pHH43 were substituted with Salmonella adhesin operon lpfABCDE and kanamycin resistance gene by homologous recombination. The reconstructive plasmid was transformed into HPSS1, and then the expresson of adhesin lpfA from the outer membrane proteins of H.pylori was verified.Conclusions:Our work explore a mutagenesis procedure designed for gene replacement in H. pylori, the results indicate that the in vivo recombination is a useful method for function analysis of H.pylori protein. A E. coli-H. pylori shuttle vector was also constructed and a heterogenous protein was successfully expressed.
        

幽门螺杆菌基因敲除方法的建立及其应用研究

摘要9-11
Abstract11-12
前言13-23
    1 幽门螺杆菌与胃部的相关疾病13-16
        1.1 幽门螺杆菌与胃炎13-14
        1.2 幽门螺杆菌与消化性溃疡14
        1.3 幽门螺杆菌与胃癌和胃MALT淋巴瘤14-16
        1.4 幽门螺杆菌与毒力因子16
    2 细菌基因突变技术的应用16
    3 幽门螺杆菌毒力致病因子研究进展16-22
        3.1 CagA17-18
        3.2 VacA18-19
        3.3 尿素酶19
        3.4 BabA19-20
        3.5 DupA20
        3.6 OipA20-21
        3.7 SabA21
        3.8 其他黏附因子21-22
    4 研究目的与意义22-23
第一部分 实验一 幽门螺杆菌基因敲除方法的建立23-32
    1. 材料和方法23-28
        1.1 实验材料23-25
            1.1.1 质粒和菌株23-24
            1.1.2 酶与试剂24
            1.1.3 培养基及溶液24-25
        1.2 实验方法25-28
            1.2.1 H.pylori的培养25
            1.2.2 H.pylori基因组的制备25
            1.2.3 引物序列设计25-26
            1.2.4 PCR扩增目的基因26
            1.2.5 打靶载体的构建26-27
            1.2.6 幽门螺杆菌高效电转化方法的建立27
            1.2.7 全菌PCR鉴定含重组质粒的转化子27-28
    2 结果28-30
        2.1 幽门螺杆菌cagA基因的敲除28-30
            2.1.1 用于cagA基因敲除的长同源臂外源DNA的构建28-29
            2.1.2 打靶载体的构建29
            2.1.3 基因的敲除及PCR鉴定29-30
    3 讨论30-32
实验二 幽门螺杆菌hp0596基因缺失突变体的构建32-40
    1 材料和方法32-34
        1.1 实验材料32
        1.2 实验方法32-34
            1.2.1 打靶载体的构建32-33
            1.2.2 电转感受态的制备及电击转化33
            1.2.3 全菌PCR鉴定含重组质粒的转化子33-34
            1.2.4 H. pylori26695(Δ0596)基因缺失突变株的RNA水平分析34
            1.2.5 H. pylori 26695Δ0596基因缺失突变株的全菌蛋白和免疫印迹分析34
    2 结果34-38
        2.1 幽门螺杆菌hp0596基因的敲除34-37
            2.1.1 打靶载体34-36
            2.1.2 hp0596基因的敲除及PCR鉴定36-37
        2.2 hp0596基因缺失突变株的RT-PCR分析37
        2.3 H. pylori26695Δ0596基因缺失突变株的全菌蛋白分析37-38
    3 讨论38-40
第二部分 利用反选择系统在幽门螺杆菌中建立无痕敲除方法40-52
    1 材料与方法40-44
        1.1 实验材料40-41
        1.2 实验方法41-44
            1.2.1 引物合成与测序41-42
            1.2.2 打靶载体的构建42
            1.2.3 全菌PCR鉴定含重组质粒的转化子42-43
            1.2.4 RNA水平的鉴定以及反转录RT-PCR的鉴定43
            1.2.5 H.pylori26695(ΔureB)基因缺失突变株的免疫印迹分析43
            1.2.6 幽门螺杆菌尿素酶活性分析43-44
    2 结果44-50
        2.1 幽门螺杆菌ureB基因敲除44-48
            2.1.1 PCR扩增体系及其打靶载体的鉴定44-47
            2.1.2 H.pylori26695ΔureB缺失突变株的PCR鉴定47-48
        2.2 H.pylori26695(ΔureB)缺失突变株的RT-PCR鉴定48-49
        2.3 H.pylori26695 ΔureB缺失突变株的免疫印迹鉴定49-50
        2.4 H.pylori26695(ΔureB)缺失突变株的尿素酶活性的分析50
    3 讨论50-52
第三部分 S.Typhimurium黏附素lpfA在H.pylori中的表达及其应用研究52-66
    1 材料与方法53-58
        1.1 实验材料53-54
            1.1.1 质粒和菌株53
            1.1.2 酶与试剂53-54
            1.1.3 培养基54
            1.1.4 溶液54
        1.2 实验方法54-58
            1.2.1 幽门螺杆菌感染的动物模型的建立54-55
            1.2.2 引物55
            1.2.3 穿梭载体的改造55-56
            1.2.4 λRed基因的诱导表达和感受态细胞的制备56
            1.2.5 电击转化56-57
            1.2.6 全菌PCR鉴定含重组质粒的转化子57
            1.2.7 氨苄青霉素抗性基因的去除57
            1.2.8 穿梭表达载体的构建57
            1.2.9 外膜蛋白的提取57-58
    2 结果58-64
        2.1 小鼠胃黏膜定植HPSS1菌株的鉴定58-59
        2.2 E.coli-H.pylori穿梭表达载体的构建59-61
            2.2.1 穿梭打靶载体的鉴定60
            2.2.2 用PCR方法鉴定含重组质粒的转化子60-61
            2.2.3 鉴定氨苄青霉素抗性基因的去除61
        2.3 穿梭表达载体的鉴定61-62
        2.4 黏附素lpfA蛋白表达62-63
        2.5 黏附素lpfA蛋白在外膜的表达63-64
    3 讨论64-66
        3.1 幽门螺杆菌感染的动物模型的建立64
        3.2 质粒的选择和构建64-65
        3.3 肠黏附素蛋白的选择和表达65-66
参考文献66-71
致谢71-72
攻读硕士学位期间参加的学术活动与发表文章72
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