Identification And Characterization Of CagL Gene From Cag Pathogenicity Island In Helicobacter Pylori

Objective:To done the cagL gene of Helicobacter pylori(H.pylori) NCTC 11637 and to perform sequencing and analysis of biological information;To study the effect of CagL of H.pylori on the expression of IL-8 mRNA in cell line GES-1,and to approach the gene function of cagL gene in typeâ…£secretion system(TFSS), which was encoded by cag pathogenicity island(cag PAI) of H.pylori,All of these will shed light on the pathogenic mechanism of H.pylori.Methods:1.Oligonudeotide primers were designed according the sequence of H.pylori 22695 in GenBank,using Primer Premier 5.0 software.H.pylori cagL gene was amplified from the genome DNA of H.pylori NCTC 11637 by Polymerase chain reaction(PCR).The purified PCR products were doned into pGEM-T vector.The sequence of cagL was analyzed through bioinformatics approach.2.The expression vector PET-28a-cagL was constructed and transformed into E.coli BL21(DE3) by molecular doning method.The protein was expressed and characterized via SDS-PAGE and Western blot methods after induced by IPTG..The target protein CagL was purified and collected by Ni²⁺-NTA cloumn.3.The rabbit was immunized by the target fusion protein combined with the Freud's complete adjuvant.The titer of purified polyclonal antibody was detected by enzyme- linked immunosorbent assay(ELISA).4.The purified CagL protein was renaturated and co-incubated with GES-1 cells.IL-8 mRNA expression was determined for the effect of concentration-dependent by reverse transcriptase polymerase chain reaction (RT-PCR).5.H.pylori was harvested and resuspended,the lysate was fractionated into Periplasmic fractions,Cytosolic fractions,inner membrane fractions and outer membrane fractions by ultracentrifuge.The subcellular localization of CagL was detected by Western blot.6.H.pylori were co-incubated with GES-1 cells for determining the translocation of CagA protein after neutralization of CagL polyclonal antibody by Western blot.Results:1.we cloned cagL gene from the H.pylori NCTC 11637 genomic DNA.DNA sequence analysis showed the sequence of cagL was 654bp,which encodes a product of 217 amino acids with a predicted molecular mass(Mr) of 32 kD.GenBank accession number is GU937872.The homology of the strains in nucleotide acid was 96%～98%.Their homogeneity in the amino acids was 97%～99%.DNAStar software predicted that it possessed good antigencity.2.The prokaryotic expression vector PET-28a-cagL was efficiently transformed into E.coli BL21(DE3).The fusion preotein was expressed in E.coli BL21(DE3) at 30℃after 1.0mmol/L IPTG induction for 4 hours.The result of SDS-PAGE and Western blot suggested that The fusion preotein is the target protein.The target protein CagL was purified and collected.3.The anti-CagL PAb was prepared by immunization rabbit with purified target fusion protein.The titer of PAb was 1:3.2×10⁵ by ELISA.4.The expression of IL-8 mRNA in GES-1 cells could be induced by CagL in a dose-dependent manner.5.The result of Western blot suggested that CagL is localized to the bacterial inner- oute membraner.The CagL polyclonal antibody can inhibit translocation of CagA protein in a dose-dependent manner.Conclusion:In this study,we have cloned the cagL gene successfully,the fusion protein CagL was obtained from E.coli.The expression of IL-8 mRNA in GES-1 cells could be induced by CagL in a dose-dependent manner.It also suggests that CagL is localized to the bacterial inner-oute membraner and the CagL polyclonal antibody can inhibit translocation of CagA protein in a dose-dependent manner.All of these will shed light on the pathogenic mechanism of H.pylori.