Establishment Of The BSH Gene From Lactobacillus Plantarum In Lactococcus Lactis NZ9000Expression Systerm

In this experiment, recombinant Lactococcus lactis NZ9000which has activity is obtained byrecombinant expression method. A pair of specific primers were designed according to thesequences of BSH gene in GenBank and the multiple clone site of the sequence of Lactobacillusexpression vector pNZ8148by primer5software for amplifing the sequence of BSH codingregion;BSH gene of L.plantarum M1-UVS29was amplified by PCR, then inserted into theLactobacillus expression vector pNZ8148, named pNZ8148-BSH. The recombinant expressionvector was transformed into Lactococcus lactis NZ9000. Target gene was induced and expressedby nisin; expressed protein was purified by electric elution method and analysed by SDS-PAGE,then identified the immunogenicity by Western-blotting. Finally according to the physicochemicalproperties of BSH determined the activity.BSH gene from L.plantarum M1-UVS29was cloned, then recombinant prokaryotic express-ion plasmid pNZ8148-BSH which carried BSH gene was constructed successfully in this study. p-NZ8148-BSH was transformed into Lactococcus lactis NZ9000by electrotransformation andanalyzed by DNA Star software shows that the homology between amino acid and sequence that itcoded was100%, then prokaryotic expression system of lactococcus lactis has been aequiredwhich can stably expressed pNZ8148-BSH. BSH had a high-level expression in recombinantLactococcus lactis NZ9000by induced with nisin for3h at30℃. SDS-PAGE indicated that thereis a target protein strap on the molecular weight of about37kDa,the same to expected result.Theexpressed protein reached a purity more than90%after purification by electroelution. The resultof western-blotting identification shows that expressed protein has good antigenicity because thereis a specific reaction between expression products and Lactobacillus plantarum polyclonalantibody.According to physicochemical properties of BSH determined the activity of expressionprotein was0.77umol. min-1, then it will lay a foundation for the further research on BSH geneengineering lactic acid bacteria and the development of the food and micro ecology preparationwhich can reduce cholesterol.