Pro-Nisin Structure Gene Cloning And Expressing In Lactococcus Lactis NZ9800

Nisin is a highly modified peptide antibiotic produced by certain strains of Lactococcus lactis. It is of great interest to the food industry because of its efficient antimicrobial activity aginst a wide range of gram-positive organisms,incluing many spoilage bacteria and food pathogens such as Listeria,Clostridium,and Bacillus species.Lactococcus lactis undergoes a long history of safe use in a variety of dairy and other food fermentations. In the recent years, the genetics of L.lactis has been greatly advanced. This has resulted in the availability of genetically modified L.lactis with prospects for application in food industry. In this studay, genomic DNA extracted from Lactococcus lactis NIZO R5 was used directly as the template for PCR in this paper. The oligonucleotide primers introduced into two restriction site Hindlll and EcoRI were designed and synthesized according to the nisA sequence reported by GenBank. A DNA fragment was amplified from Lactococcus lactis NIZO R5 genome by means of PCR and cloned into pMG36e plasmid. The recombinant plasmid was transformd into E.coliJM 109 and identified by restriction endonucleases analyzing. The recombinant plasmid was introduced into Lactococcus lactis NZ9800. SDS-PAGE analysis revealed that Lactococcus lactis NZ9800 harbouring pMG36e/nisA restored little ability of nisin production. Results suggested as following:1. The high purity of genomic DNA extracted by TriPure Isolation Reagent was observed. DNA agarose gel electrophoresis showed that the genome had ahigh integrity without degradation.And also,spectrophotometric analysis indicated that the genomic DNA had no pollution by protein and RNA.2. A approximately 460bp DNA fragment was amplified by PCR from Lactococcus lactis NIZO R5 genome.The primers used in this study comprised the following nucleotide sequences: 5' -CGCGAATTCGATATAGGTTTATTGAGT-3' and 5' -ATGAAGCTTATCCATGTCAGAACTAA-3' .Conditions used for the PCR consisted of 30 cycles of 94癈 for0.5min,45癈 for Imin and 72癈 for1.5min, plus one additional cycle of 72 for 10min.3. The amplified DNA fragment was then ligated into the Hindlll and EcoRI sites of pMG36e. The ligation mixture was transformed into JM109 for the initial cloning.The recombinant plasmid pMG36e/nisA isolated from JM109 transformants were analyzed by restriction enzyme digestion.4. The recombinant plasmid pMG36e/nisA was introduced into Lactococcus lactis NZ9800.SDS-PAGE analysis revealed that Lactococcus lactis NZ9800 harbouring pMG36e/nisA restored no ability of nisin production.