含质粒人两歧双歧杆菌的分离、质粒测序和生物信息学分析

【作者】 马永平；

【导师】 邱宗荫；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2005， 博士

【摘要】 双歧杆菌(Bifidobacteria)是人和动物肠道内的主要生理菌之一,由于它具有抑制有害菌、免疫赋活、抑癌和营养等多种生物学功效,双歧杆菌已经广泛应用于食品和保健品生产,对它的研究早已成为微生态学界和食品学界的热点,但是现有的生产菌株携带天然质粒的情况却很少见。因此,对生产菌株进行基因水平的改造显得力不从心。如果能够分离到携带天然质粒的双歧杆菌株,通过分子生物学手段对它进行基因工程改造,使双歧杆菌制品增加新的抗感染、抗肿瘤功能,或将它改造成基因治疗的给药系统等,将使双歧杆菌方面的研究和应用升华到一个新的层次。 本研究从健康男性青年粪便中分离到一株携带天然质粒的人两歧双歧杆菌株,在此基础上开展了以下几方面的研究工作: 第一部分 携带天然质粒的双歧杆菌种株分离和鉴定, 1. 在 MRS 和 BLb 培养基的基础上,结合文献进展和实验室条件,研制出了对双歧杆菌有较强选择性的培养基,命名为 ESB(enhanced selective medium for Bifidobacteria)。 2. 用改良的ESB培养基从健康男青年新鲜粪便中分离到一株携带天然质粒(命名为 pBIF10)的人源双歧杆菌,实验室编号 B200304。在 1.0%琼脂糖凝胶上,测得该质粒的相对分子质量约为 10kb。通过对该菌株的形态学观察、糖发酵试验和 16S rDNA 序列分析,证明该菌株为两歧双歧杆菌(Bifidobacterium bifidum);HPLC 法测得其 G+C mol%为 55.6。 3. 对该菌株申报了国家发明专利(公开号:1513984),目前该菌株保存在中国典型培养物保藏中心(CCTCC),菌株编号 CCTCC M203049。更多还原

【Abstract】 Bifidobacteria are natural members of the human intestinal microflora. They are thought to have a positive impact on the health of the host for its inhibiting the pathogenic micro-organism growth in intestines, enhancing the immune response, inhibiting tumor angiogenesis and improving nutritions. Due to these possible health promoting properties, products supplemented with Bifidobacterium are now widely available. It has been a focal topic in both microecology and food science (dairy) study fields. However, there is a little resident plasmid harboring in applied bifidobacteria strains. As a consequence, it is very difficult to improve the applied bifidobacteria strains in molecular level via plasmid manipulation. By contraries, it is possible to improve the applied strains via DNA manipulation if we could isolate a bifidobacterium strain harboring resident plasmid. So the bifidobacteria products would gain new traits such as enhanced health properties, improved anticancer functions, or would be constructed as a new gene transfer system in solid tumor therapy. In this study, a new bifidobacterium strain harboring a resident plasmid was isolated from a healthy man’s feces. Some fundamental studies have been done. Part one: Isolation and identification of bacterium strain 1. A enhanced selective medium for bifidobacteria (signature ESB) has been developed on basis of the bifidobacteria selective medium such as MRS and BLb. 2. A Bifidobacterium strain, lab number B200304, with a resident plasmid, signature pBIF10, was isolated. The relative molecular weight of the plasmid was about 10 kilobasepairs on 1.0% agarose gel electrophoresis. The Bifidobacterium strain was identified as B. bifidum according to its carbohydrate fermentation trials and its 16S rDNA sequence analysis. The G+C mol% value is 55.6% via HLPC (high performance liquid phase chromatography). 3. The Bifidobacterium bifidum B200304 declared a National invention patent(publish number: 1513984). The strain, serial number CCTCC M203049, now was conserved in CCTCC( the China center for type culture collection). 4. In order to appraise the safety of M203049 strain, the living bacteria have been injected into rabbit model via ear vein. The rabbits have been made as asepsis necrotic models by HIFU (high-intensity focused ultrasound). The results suggested that the living B. bifidum M203049 strain was safety for animal injected via vein and can targeted proliferation in necrotic area. Part two: The extracting and sequencing of the plasmid pBIF10 (M203049) 1. Combining digested with mutanolysin and extracted by plasmid extract kit, a B. bifidum plasmid purifying scheme has been established. 2. The plasmid pBIF10 has been sequenced and confirmed as a double strand DNA with 9275kb. Part three: Bioinformatics analysis of plasmid pBIF10 1. The plasmid pBIF10, searched by BLASTn(NCBI web net) and gfinder (softberry web net) , had been confirmed containing 13 open read frames (ORFs, or genes) at lest. Five proteins had been identified ,which shared very high orthology score with mobilization protein A and B (Mob A and Mob B),replication protein(Rep A) ,tetracycline resistanceQ(TetQ) and transposase(Tra). Besides,a conserved hypothetical protein in bacteria and two patches of tetracycline resistance element regulator (Rte A) also had been idetified. However, there were no significant conserved sequences found for other five ORFs.2. Analyzing with program EQUICKTANDEM, EINVERTED, ETANDEM, mreps and PANDROMES, several repeats had been searched in pBIF10 sequence, which maybe play an important role in gene regulation. The plasmid replication initial origin(ori,nt1087-1240),arrayed three direct repeats signature iteron, had been recognized by bioinformatics analyzing , which was very similar to sso structure of RC type plasmid. It implied that pBIF10 might be replicate in RC model. 4. Twenty one promoters had been searched by program BPROM, and the 13 ORFs searched by gfinder were all included. 5. The five proteins (RepA,Mob A,Mob B, Tra and TetQ) h更多还原