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.

Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA

AIM: To establish and assess the methods for quantitative detection of serum duck hepatitis B virus (DHBV) DNA by quantitative membrane hybridization using DHBV DNA probe labeled directly with alkaline phosphatase and fluorescence quantitative PCR (qPCR).METHODS: Probes of DHBV DNA labeled directly with alkaline phosphatase and chemiluminescent substrate CDP-star were used in this assay. DHBV DNA was detected by autoradiography,and then scanned by DNA dot-blot. In addition, three primers derived from DHBV DNA S gene were designed. Semi-nested primer was labeled by AmpliSensor. Standard curve of the positive standards of DHBV DNA was established after asymmetric preamplification, semi-nested amplification and on-line detection. Results from 100 samples detected separately by alkaline phosphatase direct-labeled DHBV DNA probe with dot-blot hybridization and digoxigeninlabeled DHBV DNA probe hybridization. Seventy samples of duck serum were tested by fluorescent qPCR and digoxigeninlabeled DHBV DNA probe in dot-blot hybridization assay and the correlation of results was analysed.RESULTS: Sensitivity of alkaline phosphatase direct-labeled DHBV DNA probe was 10 pg. The coincidence was 100% compared with digoxigenin-labeled DHBV DNA probe assay.After 30 cycles, amplification products showed two bands of about 180 bp and 70 bp by 20 g/L agarose gel electrophoresis.Concentration of amplification products was in direct proportion to the initial concentration of positive standards.The detection index was in direct proportion to the quantity of amplification products accumulated in the current cycle.The initial concentration of positive standards was in inverse proportion to the number of cycles needed for enough quantities of amplification products. Correlation coefficient of the results was (0.97, P<0.01) between fluorescent qPCR and dot-blot hybridization.CONCLUSION: Alkaline phosphatase direct-labeled DHBV DNA probe in dot-blot hybridization and fluorescent qPCR can be used as valuable means to quantify DHBV DNA in serum.

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.

Distribution and anti-HBV effects of antisense oligodeoxynu-cleotides conjugated to galactosylated poly-L-lysine

AIM: To describe distribution of the phosphorothioated antisense oligodeoxynucleotides (PS-asODNs) conjugated to galactosylated poly-L-lysine (Gal-PLL) in mice, and to observe their effects on expression of HBV gene in the 2.2.15 cells and transgenic mice.METHODS: According to the result of direct sequencing of PCR amplified products, a 16 mer phosphorothioate analogue of the antisense oligodeoxynucleotides (PS-asODNs) directed against the HBV U5-like region was conjugated to the hepatotropic Gal-PLL molecules. Its distribution was demonstrated using asODNs labeled with ^32p at the 5' terminus with a T4-polynucleotide Kinase. Its inhibition effect on HBV expression was observed in the transfected 2.2.15 cells and transgenic mice.RESULTS: The Gal-PLL and asODNs could form stable complex at a molar ratio of 2:1. As shown in the HBV-transfected 2.2.15 cells, the inhibition effects of asODNs alone and asODNs conjugated to Gal-PLL, at 10μmol/L for both, on HBsAg and HBeAg production were different,the former being 70 % and 58 %, respectively, and the latter being 96 % and 82 %, respectively. A more pronounced reduction was also observed in viral DNA load in the culture supernatant for the test with Gal-PLL-asODNs. Among many mouse organs, livers retained more asODNs molecules after administration. The preferential concentration in liver was found to be 52.14 % for Gal-PLL-asODNs, as high as 2.38-fold of that for asODNs (21.9 %). Both elements decreased gradually in liver, with 2.9 % of the former, 5.99 % of the latter retained 24 hours after the administration. The injection interval, therefore, was recommended to be 24 hours. In the transgenic mice, serum HBsAg decreased significantly (P<0.01) at the 12th day after administrating Gal-PLL- asODNs, the serum HBV DNA turned negative in 4 of the 6 mice.CONCLUSION: Antisense oligodeoxynucleotides conjugated to Gal-PLL can be concentrated in liver and intaked by hepatocytic cells. This may result in specific inhibition of expression and replication of HBV in vitro and in vivo.

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

sAIM: To examine the serological response of patients with upper gastrointestinal diseases and Helicobocter pylon (Hpy/on)infection to two Hpyloriouter membrane proteins (OMPs) (Mr18 000 and Mr26 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 NP-NTA agarose resin, colloid gold kits were prepared with purified recombinant proteins to detect H pyloH infection and H pylori-associated diseases by the immunity-marker technology. We selected 150 patients with Hpy/on'infection and digestive symptoms wibhout previous treabnent, including 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 Hpylori 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 (^13C 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 bhe two Hpy/oriOMPs,as demonstrated by the ELISA. Of the 150 serum samples from patients infected with Hpy/oH 141 (94.0%) responded positively to the recombinant protein with Mr26 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 Mr26 000 protein were 94.0%, 97.0%, and 94.5%,respe.ctively. Compared with the classic ELISA, bacteria culture and ^13C urea breath test results in detecting Hpyloriinfection, there was no significant difference (P>O.O5). For the colloid gold kit with Mr18 000, the seropositive rates were 52.0%, 40.0%, 40.0%, 53.3% and 86.7%, respectively,in Hpylori-infected palJents, and bhose wibh Hpylori-associated chronic gastritis, duodenal ulcer, gastric ulcer, and gastric cancer. There was a significant difference (P<0.05) in seropositivity between patient with gastric 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-associated gastric malignancy.