Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2.
OBJECTIVE
To construct a recombinant live attenuated Salmonella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo.


METHODS
H pylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. coli DH5alpha, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicity of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using Lipofectamine 2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 x 10(8) recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 x 10(7) CFU of live H pylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylori 4 wk post-challenge.


RESULTS
The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistent with the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that (100%) in the control group (P<0.01).


CONCLUSIONS
Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.
Xu
Can
C
Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. xxccan@sohu.com
Li
Zhao-Shen
ZS
Du
Yi-Qi
YQ
Gong
Yan-Fang
YF
Yang
Hua
H
Sun
Bo
B
Jin
Jing
J
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D004269:DNA, Bacterial / Q000235:genetics; D016481:Helicobacter Infections / Q000276:immunology / Q000517:prevention & control*; D016480:Helicobacter pylori / Q000235:genetics*; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D012480:Salmonella Infections / Q000517:prevention & control; D022562:Salmonella Vaccines / Q000235:genetics*; D012486:Salmonella typhimurium / Q000235:genetics* / Q000276:immunology; D014162:Transfection; D014510:Urease / Q000235:genetics; D014613:Vaccines, Attenuated; D019444:Vaccines, DNA / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007376:Interleukin-2; D022562:Salmonella Vaccines; D014613:Vaccines, Attenuated; D019444:Vaccines, DNA; D014510:Urease