Expression And Identification Of Urease B Subunit From Helicobacter Pylori In Lactobacillus Bulgaricus

Objective:To construct genetic engineering lactobacillus expressing Helicobacter pylori Urease B subunit and assess its safety primarily.And this work would help to develop probiotics for Hp infection as a form of probiotic preparation.Methods and Results :1. According to the ureB sequence of Helicobacter pylori in GenBank, a pair of primers was designed. And then the ureB gene was amplified from Helicobacter pylori NCTC11637 by PCR techniques. After purification, the ureB gene fragment was digested with restriction endonucleases PstI and HindIII, and then linked with the lactic acid bacterial expression vector pMG36e digested with the same enzymes to construct the recombinant plasmid pMG36e-ureB. Restriction enzyme digestion, PCR identification and DNA sequencing analysis confirmed that the construction of pMG36e-ureB was successful.2. Optimize the electroportion procedures for lactobacillus bulgaricus(L6032),And the recombinant plasmid pMG36e-ureB was transformed into L6032 by electroportion. PCR identification and restriction enzyme digestion confirmed that the recombinant plasmid pMG36e-ureB was electroporated into lactobacillus bulgaricus successful. The recombinant lactobacillus strain L6032-UreB was obtained.3. L6032-UreB was grown at 37°on MRS medium containing lactose and induced for expressing UreB. SDS-PAGE showed the genetic engineering lactobacillus could express the protein, about 64KD protein, which could react with rabbit anti-UreB serum. After subculture for 60 passages, the stability,morpy and physio-biochemical characteristic detection indicated the genetic engineering lactobacillus L6032-UreB and lactobacillus bulgaricus L6032 were exactly the same strain.Conclusion:1. The recombinant plasmid pMG36e-ureB was successfully constructed.2. The genetic engineering lactobacillus L6032-UreB expressing Hp UreB protein was constructed and identified successfully. And there is no any variation between L6032-UreB and lactobacillus bulgaricus L6032.