Research On Identification Of Bifidobacteria Producing Bile Salt Hydrolase (BSH) And Its Gene Cloning

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%.