Cloning And Sequence Analysis Of Pedicoccus Acidilactici 16SrRNA Gene And Pediocin Gene

Bacteriocin is antimicrobial peptide or protein produced by Lactic acid bacteria which attracted interest as a potential biopreservative in food systerms.A isolated strain could against Listeria monocytogenes. The strain through traditional taxonomy methods'examinate is Pediococcus acidilactici. The strain was found to produce extracellular antimicrobial substance. The antimicrobial peptide is assayed as nonorganic acid and nonhydrogen peroxide. It is sensitive to proteolytic enzymes, resistant to heat, and active over a wide range of pH. Then through appraisal of 16SrRNA sequence analysis. Designed the oligonucleotide primers according to the gene sequence reported by GenBank (X95976).Amplied the DNA fragment by means of PCR and cloned to pUCm-T Vector to sequenced.Compared the inserted DNA fraction and the Pediococcus acid 16SrRNA sequence reported, found sequence homology was 99%. Result showed the isolated strain was Pediococcus acidilactici.In order to explore P.acidilactici's antibacterial mechanism, experiment examines if this strain have the pediocin operon.,we coloned the entire operon sequence. Designed oligonucleotide primers according to the gene sequence reported by GenBank (M83924).Amplied the DNA fragment by means of PCR and cloned to pTA2 Vector.Compared the inserted DNA fraction and the sequence,and found equence homology was 99.9%.It's operon is 5595bp,made up of four genebunch, pedA , pedB, pedC,pedD,In 1688th base varied and induced one amino acid varied.The isolated strain have low output, not suits for market exploied.The experiment extracted Genomic DNA from Pediococcus acid as template. Designed the oligonucleotide primers according to the gene sequence reported by GenBank (M83924).Amplied the DNA fragment by means of PCR and cloned to pTA2 Vector. Then inserted the segment to expressing vector pET-28a.The recombinant plasmid transformed to E.coliDH5α, screened to obtained positive recombinant plasmid and transformed which to E.coli BL21(DE3),induced by IPTG ,Tricine-SDS-PAGE showed it's molecular mass as 4600Da the same as the expected molecular mass.