The development of an attenuated Salmonella typhimurium vaccine carrying plamids encoding Helicobacter pylori ureB and IL-2
Objective To construct a recombinant live attenuated Salmonella typhimurium nucleic acid vaccine encoding H.pylori ureB gene and murine interleukin 2 and to identify the immunogenicity of the proteins expressed. Methods ureB gene fragment was amplified with polymerase chain reaction (PCR) from the genomic DNA of standard H.pylori strain 17874 and murine IL 2 gene was amplified from pCIneo IL 2. ureB and IL 2 were cloned into pUCmT vector respectively. Both the genes of ureB and IL 2 were cloned into the eukaryotic expression vector pIRES through a serials of enzyme digestion and ligation reactions, the recombinant plasmids were screened by PCR reaction and restriction enzyme digestion. Then, the recombinant pIRES ureB and pIRES ureB IL 2 were transformed into LB5000 and the recombinant plasmids from LB5000 were then transformed into final host SL7207. The strains were grown in LB medium repeatedly. Recombinant pIRES ureB IL 2 and pIRES ureB were transfected into Cos 7 cells using LipofectAMINE TM 2000, the immunogenicity of UreB and IL 2 protein expressed was detected with SDS PAGE and Western blot. Results The 1700 base pair ureB gene fragment and 510 bp IL 2 were amplified and they consistent with the sequence of the H.pylori ureB and murine IL 2 respectively. It was confirmed with PCR and restriction enzyme digestion that H.pylori ureB gene and murine IL 2 were inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium nucleic acid vaccine encoding H.pylori ureB and murine IL 2 was successfully constructed and specific strip of 66kD ureB and 14kD IL 2 was detected through Western blot. Conclusion The recombinant attenuated Salmonella typhimurium nucleic acid vaccine strain encoding H.pylori ureB and immune adjuvant IL 2 with immunogenicity in vitro was constructed, it may be helpful for further investigation about the immune function of the nucleic acid vaccine in vivo .