Antibacterial Effect Of Lactobacillus Rhamnosus XA₂-14 Strain And Cloning And Expression Of Bacteriocin WO-A/2 Gene

In this thesis,Lactobacillus rhamnosus XA₂-14 strain preserved in the microbial laboratory of Inner Mongolia Agricultural University was used as the research object.Oxford cup method was used to exclude acid products and H₂O₂products from the culture medium of Lactobacillus rhamnosus XA₂-14.Protease hydrolysis test and acid-base stability test were carried out to determine the antibacterial substance produced by Lactobacillus rhamnosus XA₂-14 as bacteriocin.Then the bacteriocin WO-A/2 operon was detected at the molecular level,and the whole genome of Lactobacillus rhamnosus XA₂-14 was extracted as a template.A pair of specific primers were designed according to the full sequence of WO-A/2 gene published in Gen Bank（HV942910.1）.The target fragment was amplified from genomic DNA by PCR.After recovery and purification,the target fragment was ligated to p MD19-T vector and transformed into E.coli DH5αcompetent cells.After verification and screening,the positive clones were identified and analyzed,cloned into p ET-32a（+）expression vector and transferred into E.coli BL21（DE3）competent cells.After Amp+plate screening,plasmid double enzyme digestion and colony PCR two-step verification,the recombinant positive plasmid was successfully obtained,and the expression of E.coli BL21（DE3）was induced by IPTG.SDS-PAGE electrophoresis showed that the band was about 26k Da,which was consistent with the expected molecular weight of bacteriocin WO-A/2 protein.Subsequently,the induction conditions of bacteriocin WO-A/2 protein were optimized to determine the optimal induction time of bacteriocin WO-A/2 protein was 8 h and the concentration of IPTG was1.0 mmol/L.The bacteriocin WO-A/2 protein was purified by Ni-NTA affinity chromatography column and gradient dialysis refolding.The final purified protein concentration was 0.877±0.042 mg/m L.Then the bacteriostatic ability of bacteriocin WO-A/2 protein was tested.The results showed that the minimum inhibitory concentration was 34.93±1.28μg/m L,and the diameter of the bacteriostatic circle against Staphylococcus aureus was 18.19±0.40 mm,indicating that the bacteriocin had a good bacteriostatic effect on Staphylococcus aureus.Hemolytic test and quantitative test were carried out on the biological safety of bacteriocin WO-A/2 protein.The results showed that the bacteriocin WO-A/2 protein had certain biological safety,and it was possible to replace food preservatives and antibacterial drugs.AO/EB double fluorescence staining method was used to compare with commercial Nisin to explore its antibacterial ability and preliminarily explore the mechanism of bacteriocin WO-A/2protein.The results confirmed that the antibacterial effect of bacteriocin WO-A/2 protein on Staphylococcus aureus was achieved through the destruction of bacterial cell membrane leading to apoptosis.The results of this study provide a reference and data support for Lactobacillus rhamnosus bacteriocin to become a safe and reliable alternative.