| 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...
|
PDF Full Text Request