Identification Lmo1521 Gene Of Listeria Monocytogenes

The gram-positive Listeria monocytogenes is an ubiquitous food-borne pathogen in the environment associated with high mortality rates as 30-40%.Autolysin is a peptidoglycan hydrolases,which presents in all bacteria and involves in a variety of biological function of bacteria.At present,eight Listeria monocytogenes autolysins have been identified.Listeria monocytogenes genome sequence analysis and the transcriptome sequencing technology have revealed that lmo1521 has hypothetic amidase domain and GW modules,during the infection,detected the up-dated expression of lmo1521 gene,thus predict it may be the autolyzed gene.This experimentation proposed in this paper has verified the prediction.In this study,the CDS sequence of Listeria monocytogenes lmo1521 gene was used as a target gene to construct the pET-22b-lmo1521 recombinant plasmid,and transformed into E.coli BL21(DE3),the recombinant protein was expressed in prokaryotic expression with Isopropyl P-D-Thiogalactopyranoside(IPTG),and purified by Ni-NTA resin,Then,we get the target protein with high purity and concentration.We use a variety of methods to study the cell wall hydrolysis activity of Lmol521 protein.First,with purified cell wall of Listeria monocytogenes as a substrate,we observed whether the purified Lmo1521 protein could destroy the cell wall By using renaturing SDS-PAGE,The data was further analysed with MALDI-TOF-MS.A gram stain test using purified recombinant preotein Lmo1521 as a treatment was used to observe degradation of Listeria monocytogenes living cells,Then,based on the density of bacterial cells and the change of the cells shape,the protein's active function was analysis.The result indicated that Lmo1521 protein could degrade the cell wall of Listeria monocytogenes that produce them best in a neutral to alkaline conditions.Lmo1521 protein could not only degrade purified cell wall,but also inhibit the growth of Listeria monocytogenes living cell.Thereby this study has identified for the first time that the lmo1521 gene has autolysis function from the Listeria monocytogenes(CMC54002,serotype 1/2a)wih the experiment evidence.The Lmo1521 protein has a good bacteriostatic effect,Futher experiments verify this effect for a wide range of bacteria.It can be used for the research and development of new type of antibacterial agent and for the control of listeria disease and others.In order to further analyse the function of lmo1521 gene,we plans to build the pMAD?lmo1521 recombinant plasmid.A SOE-PCR was used to amplify lmo1520 and lmo1522 genes which locate in the upstream and downstream of target gene lmo1521,cloning to pMAD plasmid,construct pMAD A lmol521 recombinant plasmid,for the sake of pMAD ? lmo1521 recombinant plasmid and ?lmo1521 mutant strain,an antibiotic resistance gene was added between lmo1520 and lmo1522.In subsequent experiments,the pMAD ?lmo1521 recombinant plasmid will be introduced by electroporation into Listeria monocytogenes(CMC54002,serotype 1/2a),Than the ?lmol521 mutant strain will be filtered using the principle of gene knockout and the biology function of lmo1521 gene will be further studied,and these will be contribute to subsequent experiment.