Construction of prokaryotic expression system of ltB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity.
OBJECTIVE
To construct ltB-ureB fusion 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 pylori) strain Y06 and the ltB gene from Escherichia coli (E. coli) strain 44851 were linked into ltB-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. coli BL21DE3 induced by isopropylthio-beta-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 H pylori 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 binding bovine 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.


CONCLUSIONS
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 H pylori genetically engineered vaccine.
Yan
Jie
J
Department of Medical Microbiology and Parasitology, Medical College, Zhejiang University, Hangzhou 310031, Zhejiang Province, China. yanchen@mail.hz.zj.cn
Wang
Yuan
Y
Shao
Shi-He
SH
Mao
Ya-Fei
YF
Li
Hua-Wen
HW
Luo
Yi-Hui
YH
D000276:Adjuvants, Immunologic / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics*; D001428:Bacterial Vaccines / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D004768:Enterotoxins / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D016480:Helicobacter pylori / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014510:Urease / Q000235:genetics*
D000276:Adjuvants, Immunologic; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C027538:heat-labile enterotoxin, E coli; D014510:Urease