Identification And Characterization Of A Porin Protein Gene HopX In Helicobacter Pylori

Objective:To clone the outer membrane protein hopX gene of Helicobacter pylori(Hp)and to perform sequencing and analysis of biological information;To make a homologous comparison of nucleotide between NCTC11637,43504,26695 and J99;To study the effect of porin HopX of Helicobacter pylori(Hp)on the expression and secretion of IL-8 in human gastric cancer cell line SGC7901,and to determine the role of ERK1/2 mitogen-activated protein kinase(MAPK)signal pathway in IL-8 expression secretion from SGC7901 cells induced by porin HopX of Helicobacter pylori.To investigate the effects of HopX on the growth,differentiation and apoptosis of SGC7901 cells and explore its possible mechanism.Methods:1)Polymerase chain reaction(PCR)was used to amplify the hopX gene from Hp chromosomal DNA.Then the target gene was digested by restricted endonuclease enzyme of Bam HI,and inserted into the vector pGEM-T.The pGEM-T-hopX vector was transformed into E.coli DH5αand identified by restriction digestion and DNA sequencing.The recombinant vector was used to select and transform for nucleotide sequence analysis;2)The pQE30-hopX expression vector was transformed into E.coli M15.The his-HopX fusion protein was expressed in E.coli M15 with Isopropylβ-D-1- thiogalacto- pyranoside(IPTG),and identified by SDS-PAGE.The target fusion protein was purified by Ni-column,and then identified by Western Blot; 3)The rabbit was immunized by the target fusion protein combined with the Freud's complete adjuvant.The titer of purified PAb was detected by enzyme- linked immunosorbent assay(ELISA);4)HopX of Hp were purified and co-incubated with cells for determining the effect of time-dependent and concentration-dependent. LPS were used as positive control while general cells as negative control. The supernatants of samples were centrifuged and assayed for the secretion of IL-8 by ELISA.Rime time PCR was performed to study the mRNA expression level of IL-8.In addition,specialy inhibitors PD-98059 of ERK1/ERK2 in MAPK were pre-treated with cell before added HopX. Rime time PCR was also applied to determine the mRNA expression level of IL-8.after extraction RNA.;5)Cell proliferation and viability were determined by cell counting,MTT;PI binding,DNA electrophoresis and Hoechest-33258 were performed to evaluate apoptosis of SGC7901 cells.Results:1)The recombinant plasmid was constructed.DNA sequence analysis showed the sequence of hopX was 1284bp.We get a GenBank accession number EF208122.Omiga 2.0 software predicted its relative molecular mass(Mr.)was 47 kDa and possessed good antigencity.2)The prokaryotic expression vector pQE30- hopX was efficiently transformed into E.coli M15.The fusion preotein was expressed in E.coli M15 at 30℃after 1.0mmol/L IPTG induction for 4 hours.The HopX fusion protein was effectively expressed in E.coli as inclusion bodies and denaturation and refolding procedure was performed to acquire soluble protein.The fusion protein was conveniently purified using Profinity NTA-Ni-Charged Resin affinity column with above 90%purity. 3)The anti-HopX PAb was prepared by immunization rabbit with purified target fusion protein.The titer of PAb was 1:8×10~4 by ELISA method.4)The expression of IL-8mRNA and production of IL-8 in SGC7901 cells could be induced by HopX in a dose-dependent manner.The expression of IL-8 mRNA was obviously reduced after PD-98059 treatment.5)HopX can inhibit proliferation of SGC7901 cells,cannot induce apoptosis.Conclusion:A confirmed gene hopx has been prepared,the fusion protein HopX was obtained from E.coli;The expression and secretion of IL-8 in SGC7901 cells could be induced by HopX of Hp,This effect is depend on MAPK signal pathway at a certain extent.Here we show that the functional HopX may be a virulence factor which was in relation to the production of proinflamm.All thoses which facilitated further research in the function of porins and vaccine of Hp.