Antibacterial Effect Of Pediococcus Acidilactici And Its Cloning And Expression Of Bacteriocin

Pediococcus acidilactici R-4(separated and screened from Japanese organic waste disposal)preserved in the Microbiology Laboratory of Inner Mongolia Agricultural University as the research object in this paper.The acidic and H2O2 products of Pediococcus acidilactici R-4 culture solution are excluded by the Oxford Cup Tests,protease hydrolysis tests,acid-base stability tests,etc.believe that the antibacterial substances produced by the bacteria may be bacteriocins.In order to further explore the antibacterial mechanism of Pediococcus acidilactici R-4,the pediococcus operon was detected at the molecular level.In this experiment,the entire genome of Pediococcus acidilactici R-4 was used as a template,according to the pediocin PA-1 structural gene sequence released by GenBank(M83924)design and synthesize a pair of specific primers,use PCR technology to amplify the target fragment from genomic DNA,and recover and purify the target fragment to the pMD19-T vector,And transformed into E.coli DH5?.After verification and screening,the transformants identified as positive were finally analyzed and cloned into the pET-32a(+)ex-pression vector into E.coli BL21(DE3).After Amp+ plate selection,the plasmid was double digested and identified.Two-step PCR verification successfully obtained the recombinant positive plasmid,and induced the expression of competent E.coli BL21(DE3)by IPTG.After SDS-PAGE electrophoresis showed 26kDa of bands around,which was in accordance with the expected molecular weight of bacteriocin.