AIM: To construct ItB-ureBfusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacter pylori (H py...AIM: To construct ItB-ureBfusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacter pylori (H pylon) strain Y06 and the ItB gene from Escherichia coli (E. coli) strain 44851 were linked into ItB-ureB fusion gene by PCR. The fusion gene sequence was analyzed after T-A cloning. A prokaryotic recombinant expression vector pET32a inserted with ltB-ureB fusion gene (pET32a-ltB-ureB) was constructed. Expression of the recombinant LTB-UreB protein (rLTB-UreB) in E. coliBL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of Hpylori and a self-prepared rabbit anti-rUreB serum, respectively,and determine the antigenicity of the recombinant protein on inducing specific antibody in rabbits. GM1-ELISA was used to demonstrate the adjuvanticity of rLTB-UreB.Immunoreaction of rLTB-UreB to the UreB antibody positive sera from 125 gastric patients was determined by using ELISA.RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ltB-ureB fusion gene were 100%. IPTG with different dosages of 0.1-1.0 mmol/L could efficiently induce pET32a-ltB-ureB-E, coli BL21DE3 to express the rLTB-UreB.The output of the target recombinant protein expressed by pET32a-ureB-E.coli BL21DE3 was approximately 35% of the total bacterial proteins, rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori and anti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from the patients (125/125) were positive for rLTB-UreB.CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established. The expressed rLTB-UreB showed qualified immunogenicity, antigenicity and adjuvanticity. All the results mentioned above laid a firm foundation for further development of Hpylorigenetically engineered vaccine.展开更多
To construct a prokaryotic expression system of a Helicobacter pylori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays-(ELISA) for detectin.g Ca.gA.and its antibody, so as to understand ...To construct a prokaryotic expression system of a Helicobacter pylori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays-(ELISA) for detectin.g Ca.gA.and its antibody, so as to understand the manner in which the infection of CagA-expressing Hpylori(CagA+ Hpylori) isolates cause diseases.展开更多
Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It in...Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density,the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.展开更多
基金Supported by the Foundation of Ministry of Education of China forOutstanding Young Teachers
文摘AIM: To construct ItB-ureBfusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacter pylori (H pylon) strain Y06 and the ItB gene from Escherichia coli (E. coli) strain 44851 were linked into ItB-ureB fusion gene by PCR. The fusion gene sequence was analyzed after T-A cloning. A prokaryotic recombinant expression vector pET32a inserted with ltB-ureB fusion gene (pET32a-ltB-ureB) was constructed. Expression of the recombinant LTB-UreB protein (rLTB-UreB) in E. coliBL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of Hpylori and a self-prepared rabbit anti-rUreB serum, respectively,and determine the antigenicity of the recombinant protein on inducing specific antibody in rabbits. GM1-ELISA was used to demonstrate the adjuvanticity of rLTB-UreB.Immunoreaction of rLTB-UreB to the UreB antibody positive sera from 125 gastric patients was determined by using ELISA.RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ltB-ureB fusion gene were 100%. IPTG with different dosages of 0.1-1.0 mmol/L could efficiently induce pET32a-ltB-ureB-E, coli BL21DE3 to express the rLTB-UreB.The output of the target recombinant protein expressed by pET32a-ureB-E.coli BL21DE3 was approximately 35% of the total bacterial proteins, rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori and anti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from the patients (125/125) were positive for rLTB-UreB.CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established. The expressed rLTB-UreB showed qualified immunogenicity, antigenicity and adjuvanticity. All the results mentioned above laid a firm foundation for further development of Hpylorigenetically engineered vaccine.
基金Supported by the Outstanding Young Teacher Fund of Education Ministry of China and the General Research Plan of Zhejiang Provincial Science and Technology Commission,No.001110438
文摘To construct a prokaryotic expression system of a Helicobacter pylori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays-(ELISA) for detectin.g Ca.gA.and its antibody, so as to understand the manner in which the infection of CagA-expressing Hpylori(CagA+ Hpylori) isolates cause diseases.
基金Supported by the Research Plan of Jiangsu Provincial Technology Commission, No. BS2004021Advanced Talent Research Plan of Jiangsu University, No. JDG2004008
文摘Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density,the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.