【作者】 王丹；

【导师】 刘景圣；

【作者基本信息】 吉林农业大学 ， 食品科学， 2011， 硕士

【摘要】 胆盐水解酶(Bile Salt Hydrolase, BSH)是益生菌在生长繁殖的过程中分泌的一种代谢产物。该酶能将胃肠道内的结合胆盐水解,形成氨基酸以及溶解性较低的游离胆酸,而游离胆酸与人体内的胆固醇相结合产生沉淀,从而在一定程度上降低人体血清中胆固醇的含量,进而减少人类心血管疾病的发病几率。本文首先对双歧杆菌产生胆盐水解酶进行鉴定,然后采用16SrRNA分子生物学技术对该菌进行了分类鉴定,并对其胆盐水解酶基因进行克隆与序列分析,具体研究内容如下：1.本试验将双歧杆菌接种到改良的TPY培养基中进行培养,经过对该菌产生的胆盐水解酶进行定性与定量的分析,结果表明双歧杆菌产生具有一定活力的胆盐水解酶,为胆盐水解酶基因的研究提供可参考的理论依据。2.采用16SrRNA分子生物学技术对双歧杆菌进行分类鉴定,将扩增的PCR产物进行回收纯化,测序后该菌的16SrRNA为1488bp,在NCBI上进行在线BLAST比对,得知该菌与两歧双歧杆菌的同源性达到99%,确定试验室保藏的双歧杆菌为两歧双歧杆菌。3.根据菌种鉴定的结果得知与其亲缘关系最近的菌种,通过其菌种的胆盐水解酶基因序列设计—对引物,以两歧双歧杆菌的基因组DNA为模板进行PCR扩增,回收产物与pMD19-T载体进行连接,将重组克隆质粒pMD19-BSH转入到大肠杆菌JM109感受态细胞中,筛选出阳性的重组菌,对重组克隆质粒进行PCR鉴定与酶切鉴定后,将重组克隆菌进行基因的测序,胆盐水解酶基因大小为951bp。测序结果在NCBI上进行在线BLAST比对,目的基因的序列与NCBI上公布的胆盐水解酶基因的同源性达到99%,应用DNAMAN软件翻译成317个氨基酸序列后,进行比对得知同源性达到99%,表明胆盐水解酶基因克隆成功。更多还原

【Abstract】 Bile Salt Hydrolase(BSH) was metabolite of lactobacillus secreted in the process of its growth and breeding. Amino acid and free cholic of lower dissolved acid were obtained after the enzyme’s combination and hydrolysis of the bile salt in the gastrointestinal tract. To some extent, it can reduce the cholesterol level in the body since free bile acid combined with the cholesterol in the form of precipitation.Bifidobacteria producing bile salt hydrolase (BSH) was identified and then classified by molecuLar technology of 16SrRNA. Its bile salt hydrolase gene cloning and sequence was analyzed. The specific experimental contents were as follows:1. Bifidobacteria was inocuLated in the modified medium of TPY and cuLtured for a certain period. And then the enzyme produced by the strain was qualitatively and quantitatively analyzed. ResuLts showed that Bifidobacteria can produce certain activity of BSH, and give some theoretical basis for the research of BSH.2. Bifidobacteria was classified by molecuLar technology of 16SrRNA and its amplified PCR products were purified.16SrRNA of the strain sequenced was 1488bp, homology between the strain and Bifidobacteria by means of BLAST in NCBI online. And Bifidobacteria preserved in the laboratory was determined as bifid bacterium bifidum.3. With the genomic.DNA of bifidobacterium bifidum as the template, according tothe published in the Bifidobacterium bifidum strain ATCC 15696 bile salt hydrolase(bsh) gene, a pair of primes were designed by application Primer 5.0 program. Amplified by PCR, the target gene of 951bp was obtained. Combine the recovered PCR products with pMD19-T vector, the recombinant pMD19-BSH was transferred into the competent cell of E.coli JM109, and positive recombinant clone plasmid was screened. Then it was identified by PCR and restriction enzyme analysis, the recombinant vector was sequenced by Shanghai Sangon Bioengineering Companies. Compared with BLAST in NCBI online, the resuLts showed that the nucleotide homology between the sequence of target gene and BSH gene published in the NCBI attained 99%, and the amino acid homology in the NCBI attained 99%.更多还原