Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA.
OBJECTIVE
To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity.


METHODS
Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli DH5alpha, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot.


RESULTS
The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot.


CONCLUSIONS
The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.
Xu
Can
C
Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. xucan9@hotmail.com
Li
Zhao-Shen
ZS
Du
Yi-Qi
YQ
Tu
Zhen-Xing
ZX
Gong
Yan-Fang
YF
Jin
Jing
J
Wu
Hong-Yu
HY
Xu
Guo-Ming
GM
D018829:Adhesins, Bacterial; D000818:Animals; D001428:Bacterial Vaccines / Q000235:genetics; D019556:COS Cells; D003001:Cloning, Molecular; D016480:Helicobacter pylori / Q000235:genetics* / Q000276:immunology; D006388:Hemagglutinins / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012486:Salmonella typhimurium / Q000235:genetics* / Q000276:immunology; D014613:Vaccines, Attenuated / Q000235:genetics*; D019444:Vaccines, DNA / Q000235:genetics*
D018829:Adhesins, Bacterial; D001428:Bacterial Vaccines; D006388:Hemagglutinins; D011994:Recombinant Proteins; D014613:Vaccines, Attenuated; D019444:Vaccines, DNA; C095977:adhesin A, Helicobacter