Biological Function Of Hpsh0459 Localized In The Plasticity Zone Of Helicobacter Pylori

Objectivehpsh0459 in the plasticity zone of H. pylori, shares a high homology with the virB10 gene, which is predicted to form a new T4 SS with other vir genes(virB4, virB8, virB9, virB11, virD4, and virD2) called as tfs3 a. Few studies had shown the function of hpsh0459, which was explored using the immunology, molecular biology and other methods in this research. The aim of this study was to determine the biological function of hpsh0459 of H. pylori. Methods1. H. pylori WH21 was cultured at 37°C under microaerobic conditions on Karmali campylobacter agar base plates containing 8% sheep blood. After the genome DNA was extracted, the sequence of hpsh0459 was amplified using of the specific primers. The amplified target DNA of hpsh0459 was cloned into the PMD®18-T vector and sequenced by Life Technologies Corporation. After the amino acid sequence of Hpsh0459 was deduced, the bioinformatics and computational biology were analyzed by bioinformatics analysis methods.2. After the recombinant expression vector pET28a(+)-hpsh0459 was transformed into E. coli Rosetta(DE3), the recombinant Hpsh0459 fusion protein was induced by IPTG and purified using Ni-nitrilotriacetic acid resin. The protein was confirmed by Western blot with anti-His6 MAb. The polyclonal antibody of Hpsh0459 was produced by immunizing rabbit, and the titer of polyclonal antibody was detected by enzyme-linked immunosorbent assays(ELISA).3. The cytotoxicity assay and IL-8 production of Hpsh0459 were analyzed after co-cultured with GES-1 Cells using MTT and ELISA.4. H. pylori strain WH-21 strain was separated into the periplasm, cytoplasm and membrane components by ultracentrifugation, and each fraction was analyzed to determine the subcellular localization of Hpsh0459 using anti-Hpsh0459 antibody by immunoblot analysis. After the total bacterial protein of H. pylori WH-21 strain and anti-Hpsh0459 antibody were incubated, the interaction proteins of Hpsh0459 were investigated by using the co-Immunoprecipitation assay and mass spectrometry.5. A suicide vector named pBluescript-△hpsh0459::Kan was constructed for an isogenic mutant of H. pylori hpsh0459. Results1. The hpsh0459 gene in H. pylori WH-21 strain is 1233 bp consisting of 411 amino acids with 46431.4Da, which belongs to Trb I superfamily. Hpsh0459 is a hydrophilic protein, and the signal peptide was not calculated by SignalP 4.1. The potential antigen epitopes are mainly distributed at the C-terminal, and it is more favorable to preparing the antibodies by using the C-terminal of the Hpsh0459. The result of three-dimensional structure shows that Hpsh0459 is a channel structure and might involved in biological macromolecules uptake or transfer.2. The pET28a-hpsh0459(633bp) was created successful and highly expressed in E. coli Rosetta(DE3). The target protein was purified by Ni-nitrilotriacetic acid resin system. After the rabbit was immunized with purified protein, the result of ELISA indicated that the titer of the antisera was 1:32,000.3. The results of MTT assay showed that the inhibition ratio of GES-1 cells cultured with Hpsh0459(0.1μ g/mL) was significantly lower than that with Hpsh0459(20μ g/m L)5. The suicide vector pBlueKM40-Δhpsh0459::kan was constructed successfully. Conclusion:1. Hpsh0459 is a hydrophilic protein and highly homologous with the VirB10 and TrbI family proteins. Hpsh0459 is a channel structure, which might form a new type of T4 SS with other vir genes involving in DNA uptake or DNA transfer.2. The expression vector pET28a-hpsh0459 was constructed, and Hpsh0459 protein was obtained. After the rabbit was immunized with purified protein, the high titer polyclonal antibody of Hpsh0459 was produced.3. Hpsh0459 localizes in the bacterial membrane, partially in the periplasm. It interacts with the zinc finger protein and methionine ABC transporter ATP-binding protein, which might involve in DNA uptake or DNA transfer.4. Hpsh0459 has cytotoxicity with a concentration dependent manner by MTT assay, which is related with IL-8 secretion from gastric mucosal epithelial cells.5. The suicide vector pBlueKM40-△hpsh0459::kan was successfully constructed.