H.pylori尿素酶B亚单位在保加利亚乳杆菌的表达与鉴定

目的构建表达幽门螺杆菌（Helicobacter pylori，H、pylori）尿素酶B亚单位（UreB）的基因工程乳杆菌，并对其进行初步的安全性评估。方法采用高保真PCR从H.pylori标准菌株NCTC 11637中扩增ureB基因，插入乳酸菌表达质粒pMG36e，将重组质粒电转入保加利亚乳杆菌L6032中，获得表达ureB的基因工程乳杆菌。在含乳糖的MRS培养基诱导目的蛋白表达，Western blot鉴定其免疫原性。连续传代培养60代，检测基因工程乳杆菌的稳定性、形态学与生理生化特性以进行初步的安全性评估。结果特异PCR、酶切和测序鉴定均证实ureB基因克隆入表达载体pMG36e，SDS-PAGE结果显示，重组质粒pMG36e-ureB电转入保加利亚乳杆菌所构建的基因工程乳杆菌能表达约64KD的蛋白，Western blot证明该蛋白能与抗H.priori ureB的兔血清反应。稳定性、形态学与生理生化特性检测结果表明，基因工程乳杆菌与原始菌株保加利亚乳杆菌完全一致。结论成功构建能表达H.pylori UreB的保加利亚乳杆菌L6032-UreB，该基因工程菌在形态与生理生化特性上未发生任何变异，从而为探索幽门螺杆菌感染的益生菌制剂调理疗法奠定了坚实的基础。

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

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. pylor/ 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.