Construction And Characterization Of Recombinant Attenuated Salmonella Typhimurium Expressing BabA2/UreI Fusion Gene Of Helicobacter Pylori

Background and Objective:In countries, especially those with low socio-economic status, the prevalence of H. pylori infection is high, which is associated with specific gastric disease. So vaccination trials exploiting the antigenic properties of some proteins, e.g. urease, the vacuolating toxin (VacA), the cytotoxin-associated antigen CagA or blood-group-antigen-binding adhesin (BabA), is in deep understanding, in which one of the most important is attenuated bacterium expressing gene. The attenuated Salmonella typhimurium strain has low pathogenicity and satisfying invasiveness, which can directly transport the expressing vector into mammal cells to express corresponding proteins and induce specific immune response. But it can not be successfully used in human although it has been proved effective in vitro, as the plasmid contains antibiotic resistance genes. The balanced lethal system constructed by Nakayama et al. has solved the problem. In this study, the bivalence DNA vaccine of BabA2/UreI fusion gene of H. pylori was constructed. First the recombinant prokaryotic expressing plasmid containing asd gene was constructed through gene engineering, then the attenuated Salmonella typhimurium x8501 (pYA3342/ BabA2/UreI) strain expressing recombinant BabA2/UreI fusion protein was obtained by asd balanced lethal system of Salmonella typhimurium and the expression of protein was detected. This provided a basis of follow-up DNA vaccine studies of H. pylori infection.Methods:1. Cloning of BabA2/UreI fusion gene. BabA2/UreI fusion gene was amplified from pcDNA3.1(-)/BabA2/UreI eukaryotic expressing plasmid by PCR, and then the PCR product was cloned into pCF-T vector, which was transformed into Escherichia coli DH5αby using TSS. Subsequently, the T-A colonies were selected by ampicillin and X-gal, and sequenced. Afterwards, Escherichia coli DH5αwhich contained BabA2/UreI fusion gene were amplified, and the plasmid was purified and digested with SmaI and SalI. Then the fusion gene of high concentration was obtained.2. Construction of prokaryotic expressing vector. BabA2/UreI fusion gene was cloned into expression vector pYA3342 containing asd gene through SmaI and SalI double digestion. The recombinant vector pYA3342/BabA2/UreI was transformed into Escherichia coli x6212 by using TSS. The recon was selected, sequenced and endonuclease digested.3. Construction of attenuated Salmonella typhimurium x8501 expressing BabA2/UreI fusion gene. The recombinant vectors were transformed into final host attenuated Salmonella typhimurium x8501 (hisGΔcrp-28ΔasdA16) strain which was a balanced lethal recombinant by electroporation. The recombinant was identified by dual- endonuclease digestion and sequencing.4. Expression of x8501(pYA3342/BabA2/UreI) was confirmed by RT-PCR and Western Blot analysis using polyclonal anti-H. pylori antibodies from rabbit.5. Stability of the recombinant x8501(pYA3342/BabA2/UreI) was determined after incubating for five days in vitro.Results:1. BabA2/UreI fusion gene was amplified from pcDNA3.1(-)/BabA2/UreI eukaryotic expressing plasmid, base number of which identified by agarose gel electrophoresis was in accordance with anticipation. And the recombinant plasmid pCF- T/BabA2 /UreI was successfully constructed, which was confirmed by endonuclease digestion and sequencing.2. The recombinant plasmid pYA3342/BabA2/UreI was successfully constructed. The obtained sequence was completed and the length of inserted gene was 2860 bp, as showed by DNA sequencing. Furthermore, sequences of nucleotide and their coding protein sequences had high homogenicity with the sequences published by Genbank.3. Attenuated Salmonella typhimurium x8501(pYA3342/BabA2/UreI) was construc- ted successfully. And results of dual-endonuclease digestion and PCR amplification showed that recombinant plasmid pYA3342/BabA2/UreI gene had been transformed into Salmonella typhimurium x8501.4. RT-PCR confirmed that fusion gene could be effectively transcribed. Western Blot showed that the attenuated Salmonella typhimurium x8501(pYA3342/BabA2/UreI) could express fusion protein BabA2/UreI, molecular weight of 106 kDa, which could be specially recognized by polyclonal anti-H. pylori antibodies from rabbit. 5. Recombinant plasmid can stably reside in host bacteria, without lost.Conclusion:1. The recombinant plasmid pYA3342/BabA2/UreI was successfully constructed.2. The attenuated Salmonella typhimurium x8501(pYA3342/BabA2/UreI) was const- ructed successfully. The fusion gene could effectively transcribed, the fusion protein could recognize by polyclonal anti-H. pylori antibodies from rabbit, and the recombinant plasmid could stably reside in host bacteria.3. It provides a basis of important target antigen and carrier system for anti-H.pylori vaccine for human, and basis of follow-up identification of the function of protein and animal researches.