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Expression and identification of urease B subunit in L.delbrueckii subsp.bulgaricus

LI Yue,ZHANG De-chun(Key Laboratory of Laboratory Medical Diagnostics,Ministry of Education,Office of Pathogenic Biology,Chongqing Medical University,Chongqing 400016,China)  
Objective To construct genetic engineering lactobacillus expressing Helicobacter pylori(H.pylori) urease B subunit and assess its safety.Methods The ureB gene were amplified from H.pylori NCTC 11637 by PCR and cloned into lactic acid bacterial expression vector pMG36e.The recombinant plasmid was electroporated into L.delbrueckii subsp.bulgaricus L6032,and got the genetic engineering lactobacillus expressing H.pylori UreB protein.The recombinant lactobacillus strain was grown at 37 ℃ on MRS medium containing lactose and induced for expression.Western blot was applied to determine its immunoreactivity.Its stability,morpy and physio-biochemical characteristic were detected after subculture for 60 passages,in order to assess its safety.Results The ureB gene could be amplified from the recombinant expression plasmid pMG36e-ureB by PCR.The restriction analysis and sequence analysis also could prove ureB gene were cloned into expression vector pMG36e.SDS-PAGE showed the genetic engineering lactobacillus could express the protein,about 64 KD protein,which could react with rabbit anti-ureB serum.The stability,morpy and physio-biochemical characteristic detection indicated the genetic engineering lactobacillus L6032-ureB and L.delbrueckii subsp.bulgaricus L6032 were exactly the same strain.Conclusions The genetic engineering lactobacillus expressing H.pylori UreB protein is constructed and identified successfully.And there is no any variation between the genetic engineering bacteria L6032-UreB and L.delbrueckii subsp.bulgaricus L6032.This study will help to develop probiotics for H.pylori infection as a form of probiotic preparation.
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