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Construction and characterization of bivalent vaccine candidate expressing HspA and M(r)18,000 OMP from Helicobacter pylori.

ABSTRACT: AIM:To construct a recombinant vector which can express outer membrane protein (OMP) with M(r)18,000 and heat shock protein A (HspA) from Helicobacter pylori (H. pylori) in E. coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection. METHODS:The target gene of HspA was amplified from H. pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn I, BamH I simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp(18), digested by restrictive endonuclease enzyme Hind III and BamH I simultaneously. pET32a(+)/HspA and Omp(18) were recovered from 1 % agarose gel by gel kit, and ligated with T(4) ligase by BamH I digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp(18) was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot. RESULTS:Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al, there were 1.3 % and 1.4 % differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (M(r)) of the expressed product was M(r) 51,000, M(r) of protein expressed by pET32a (+) was about M(r) 20,000, and soluble expression product accounted for 18.96 % of total bacterial protein. After purification with Ni(+2)-NTA agarose resins, the purification of recombinant fusion protein was about 95 %. Western blot showed that recombinant fusion protein could be recognized by the patients' serum infected with H. pylori and anti-Omp(18) monoclone, suggesting that this protein had good antigenicity. CONCLUSION:The gene coding for H. pylori M(r)18,000 OMP and HspA was cloned and expressed successfully. The results obtained lay the foundation for development of H. pylori protein vaccine and a quick diagnostic kit.

SUBMITTER: Jiang Z

PROVIDER: S-EPMC4611538 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Construction and characterization of bivalent vaccine candidate expressing HspA and M(r)18,000 OMP from Helicobacter pylori.

Jiang Zheng Z Huang Ai-Long AL Tao Xiao-Hong XH Wang Pi-Long PL

World journal of gastroenterology 20030801 8

<h4>Aim</h4>To construct a recombinant vector which can express outer membrane protein (OMP) with M(r)18,000 and heat shock protein A (HspA) from Helicobacter pylori (H. pylori) in E. coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection.<h4>Methods</h4>The target gene of HspA was amplified from H. pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn I, Bam ...[more]

PMID: 12918115

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Project description:BACKGROUND:Helicobacter pylori (H. pylori) infect more than half of the world population, and they cause different serious diseases such as gastric carcinomas. This study aims to design a vaccine on the basis of cagW against H. pylori infection. After pcDNA3.1 (+)-cagW-CS-NPs complex is produced, it will be administered into the muscles of healthy BALB/c mice in order to study the effect of this DNA vaccine on the interleukin status of mice, representing its effect on the immune system. After that, the results will be compared with the control groups comprising the administration of cagW-pCDNA3.1 (+) vaccine, the administration of chitosan and the administration of PBS in the muscles of mice. METHODS:The cagW gene of H. pylori was amplified by employing PCR, whose product was then cloned into the pcDNA3.1 (+) vector, and this cloning was confirmed by PCR and BamHI/EcoRV restriction enzyme digestion. CagW gene DNA vaccine was encapsulated in chitosan nanoparticles (pcDNA3.1 (+)-cagW-CS-NPs) using a complex coacervation method. The stability and in vitro expression of chitosan nanoparticles were studied by DNase I digestion and transfection, and the immune responses elicited in specific pathogen-free (SPF) mice by the pcDNA3.1 (+)-cagW-CS-NPs were evaluated. Apart from that, the protective potential pcDNA3.1 (+)-cagW-CS-NPs was evaluated by challenging with H. pylori. RESULTS:The pcDNA3.1 (+)-cagW-CS-NPs comprises cagW gene of H. pylori that is encapsulated in chitosan nanoparticles, produced with good morphology, high stability, a mean diameter of 117.7 nm, and a zeta potential of + 5.64 mV. Moreover, it was confirmed that chitosan encapsulation protects the DNA plasmid from DNase I digestion, and the immunofluorescence assay showed that the cagW gene could express in HDF cells and maintain good bioactivity at the same time. In comparison to the mice immunized with the control plasmid, in vivo immunization revealed that mice immunized with pcDNA3.1 (+)-cagW-NPs showed better immune responses and prolonged release of the plasmid DNA. CONCLUSIONS:This research proves chitosan-DNA nanoparticles as potent immunization candidates against H. pylori infection and paves the way for further developments in novel vaccines encapsulated in chitosan nanoparticles.

Dataset Information

Construction and characterization of bivalent vaccine candidate expressing HspA and M(r)18,000 OMP from Helicobacter pylori.

Publications

Construction and characterization of bivalent vaccine candidate expressing HspA and M(r)18,000 OMP from Helicobacter pylori.

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