Construction and characterization of bivalent vaccine candidate expressing HspA and M_r18 000 OMP from Helicobacterpylori

AIM: To construct a recombinant vector which can express outer membrane protein (OMP) with Mr18 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 (+)/Omp18, digested by restrictive endonuclease enzyme Hind Ⅲ and BamH Ⅰ simultaneously. pET32a(+)/HspA and Omp18 were recovered from 1% agarose gel by gel kit, and ligated with T4 ligase by BarmH Ⅰ digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp18 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 (Mr) of the expressed product was Mr 51 000,Mr of protein expressed by pET32a (+) was about Mr 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-Omp18 monoclone, suggesting that this protein had good antigenicity.CONCLUSION: The gene coding for H. pylori Mr18 000OMP 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.

Cloning and sequence analysis of gene oipA encoding an outer membrane protein of human Helicobacter pylori

AIM: To construct a recombinant E. col/strain that would highly express the proinflammatory outer membrane protein of human Helicobacter pylori ( H pylon).METHODS: The oipA DNA was amplified by PCR, inserted into pET-32a, and transformed into Top10 E. col/strain. This recombinant plasmid of Top10 was sent out for nucleotide sequence analysis. Finally this sequence AF479754 was compared with HP0638 and JHP0581.RESULTS: The sequence of the aim gene was obtained. It had 924 base pairs. The identity was 95.32% against HP0638, 95.02% against JHP0581, which was higher than the identity between HP0638 and JHP0581.CONCLUSION: Although the aim gene was obtained, but it was different from the published sequence of GenBank. It is not clear what makes this difference. Maybe it is because different strain was used or because there were some variations. So more researches are required to prove it.

Diagnosis of Helicobacterpyloriinfection and diseases associated with Helicobacter pylori by Helicobacter pylori outer membrane proteins

AIM:TO examine the serological response of patients with upper gastrointestinal diseases and Helicobocter pylori(H pylon) infection to two Hpyloriouter membrane proteins(OMPs) (M,18 000 and M,26 000)acquired by gene recombinant technique,and to determine the diagnostic significance of serological tests derived from these OMPs. METHODS:Recombinant vectors encoding the two Hpylori OMPs were used to transform and express in BL21(DE3) E.COli.After purification with Ni^(2+)-NTA agarose resin,colloid gold kits were prepared with purified recombinant proteins to detect H pylori infectJon and H pylori-associated diseases by the immunity-marker technology.We selected 150 patients with Hpyloriinfection and digestive symptoms without previous treatment,induding chronic gastritis(n=60),duodenal ulcer (n=30),gastric ulcer(n=30),and gastric cancer(n=30). As controls,33 Hpylori-negative healthy volunteers were also recruited.Serum samples were collected from all subjects,and the antibodies to specific proteins of H pylori were tested with the colloid gold test kits.The sensitivity, specificity and accuracy of the colloid gold tests were evaluated,by using the combination of standard diagnostic methods(^(13)C urea breath test and bacteria culture)and classic enzyme-linked immunosorbent assay(ELISA)as reference. RESULTS:After purification with Ni^(2+)-NTA agarose resin, the purity of recombinant fusion proteins was about 95%. The recombinant fusion proteins were recognized by the specific monodonal antibodies against the two Hpylori OMPs, as demonstrated by the ELISA.Of the 150 serum samples from patients infected with Hpylori 141(94.0%)responded positively to the recombinant protein with M_126 000,while the seropositive rates were 95.0%,96.7%,96.7% and 90.0% for patients with H pylori-associated chronic gastritis, duodenal ulcer,gastric ulcer,and gastric cancer respectively. The sensitivity,specificity,and accuracy of the colloid gold kit with M_1 26 000 protein were 94.0%,97.0%,and 94.5%, respectively.Compared with the classic ELISA,bacteria culture and ^(13)C urea breath test results in detecting Hpylori- infection,there was no significant difference(P>0.05).For the colloid gold kit with M,18 000,the seropositive rates were 52.0%,40.0%,40.0%,53.3% and 86.7%,respectively, in Hpylori-infected patients,and those with Hpylori-assodated chronic gastritis,duodenal ulcer,gastric ulcer,and gastric cancer.There was a significant difference(P<0.05)in seropositivity between patient with gastdc cancer(86.7%) and those with other diseases(43.3%). CONCLUSION:The two colloid gold kits derived from the recombinant OMPs are useful tools either for detecting Hpyloriinfection,or for,predicting Hpylori-assodated gastric malignancy.