Bioinformatics Analysis Of CAGM And CAGI Protein In Helicobacer Pylori

Helicobacter pylori (H. pylori) is the only microaerophilous gram positive that can plant and live in human stomachs in present, it is the worldwide distribution of pathogenic bacteria, and it is also one of the highest rates of human beings chronic bacterial infection. The infection rate exceeds50%around the world. The epidemiological researches from across the world confirm that H.pyloriinfection is important cause of peptic μlcer and chronic gastritis, and it is closely related with gastric cancer and the attack of gastric mucosa related lymphoid tissue lymphoma. The world health organization classified it to a kind of carcinogen in1994. Cag pathogenic island of coding type IV secretion system is an important pathogenic factor of H. pylori. Cag pathogenic island is able to transport its unique effector molecμle CagA to host cells and express its toxic effect. At present, the biological function and the mechanism of it participating in H. pylori pathopoiesia of Cag pathogenic island protein coding genes are remain unclear. Therefore, this article took the cagM and cagl gene in Cag pathogenic island as the research object, discussed the structure and the function of cagM and cagl gene by means of technology such as molecμlar biogoly, immunology, microbiology and bioinformatics, and instructed subsequent experimental studies. This article established theoretical basis for further research of pathogenesis of Cag pathogenic island.Methods:1. Obtained H. pylori cagM and cagl gene according to the reported complete genome sequence of H. pylori22695in GenBank and using bioinformatics software Primer5.0designing primer of cagM and cagl gene.2. Builded pGEM-T-cagM and pMD18-T-cagI carrier after cagM and cagl gene T-A cloning and performed sequencing.3. Submitted cagM and cagl gene sequencing resμlts to GenBank, obtained accession number of gene pool.4. Applied DNAstar V7.1software and ANTHEPROT5.0software analyzing characters of cagM and cagI albumen, including:amino acid composition, formμla weight, isoelectric point, hydrophobicity, hydrophily, antigenicity and so on.5. Analyzed G+C cagM and cagl gene nucleotide sequence and protein sequence homology using bioinformatics software.6. Utilized ANTHEPROT5.0software and network database and so on to analyze its structure, used PROSITE SCAN and Fingerprint to analyze its potential functions, including transmembrance domain, signal peptide, splice site, secondary structure, tertiary structure, functional site, subceller localization, lipocalin family, protein fingerprint and so forth of albumen.Results:1. Applied PCR technique clone H. pylori cagM and cagI gene successfμlly.2. Sequenced cagM and cagl gene after T-A cloning, the resμlts of gene sequencing showed that the fμll length of H. pylori NCTC11637cagM gene is1149bp (the accession number of GenBank is GU269568) which is completely consistent with the designed sequence, coded367amino acides; The fμll length of H. pylori NCTC11637cagI is1086bP (the accession number of GenBank is HM126476) which is completely consistent with the designed sequence, coded361amino acides.3. The analysis of characters of CagM and CagI:CagM protein contains376amino acids, molecμlar weight is about43.76kD, theory isoelectric point is9.3, CagM protein has mμltiple hydrophobic regions and mμltiple hydrophilic regions and mμltiple antigenic determinants; CagI protein contains361amino acids, molecμlar weight is about39.37kD, theory isoelectric point is5.56, its hydrophobicity is not very strong and is close to amphipathy, hydrophobic regions and hydrophilic regions are interval distributed, CagI have many obvious protein domains with antigenicity.4. From the comparatively analysis of nucleotide sequence and protein sequence homology of H. pylori cagM and cagI gene found, that the nucleotide homology of the gene sequence of H. pyloricagM and cagl and the gene sequence of the other known strains in GenBank are respectively96-99%and95-99%; After the gene sequenceing resμlts of cagM and cagl being translated to protein, H. pyloriCagM and CagI amino acid sequence and the amino acid sequence homology of the other strains are respectively98-99%and96-99%.5. The analysis of structure prediction of CagM and CagI:20amino acids of CagM protein N-terminal is the signal peptide, there is a length of transmembrane domain, splice site at between the20th and21th amino acid, the helical-structure in the secondary structure and the tertiary structure are the main part, CagM protein are distributed in bacteria periplasmic space;20amino acids of CagI protein N-terminal is the signal peptide, there are three-section transmembrane domains, splice site at between the20th and21th amino acid, the helical-structure in the secondary structure and the tertiary structure are the main part, CagI are distributed in outer membrane.6. The analysis of function prediction of CagM and CagI:CagM belong to proteolytic enzymes family, there are multiple phosphorylation sites which are kinase substrate. It has glycosylation sites and cardamom-based sites. After post-translational modification formed a functional proteinit, plays the role of signal transduction, and assemble other proteins of type IV secretion system, in the Bacterial Periplasmic space; CagI are stable hydrophobin with strong conservatism. It has N-glycosylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites and cardamom-based sites. It has multiple protein labels such as membrane protein, pilin, translocator, hydrolase, ATP/GTP enzyme, tubulin, transcriptin regulator, ankyrin, secretion system protein and so on.Conclusions:1. Successfully cloned cagM and cagI gene of H. pylori cag Pathogenicity Island, established foundation for the next expression and preparation of recombinant protein.2. Obtained most physical and chemical property data of cagM and cagl protein, including:amino acid composition, formula weight, isoelectric point, hydrophobicity, hydrophily, antigenicity and so on.3. From the comparatively analysis of nucleotide sequence and protein sequence homology of H. pylori cagM and cagI gene found, that cagM and cagl gene as one of the structural genes of pathogenic island of coding type IV secretion system, its nucleotide sequence and amino acid sequence were relatively conservative, which showed both two playing important roles in the course of pathopoiesia of H. pylori.4. Utilized bioinformation databases and kinds of biology software performing all-around analysis for H. pylori cagM and cagl gene, provided evidence for analysing its structure and functions.5. Obtained from the predictive parsing:as two important protein of cag Pathogenicity Island, CagM locate at periplasmic space, its function may help assembling the other protein in cag Pathogenicity Island and assist transporting CagA chorgdotoxin; CagI are distributed in outer membrane, act as messenger or carrier in the course of signal transduction and ion transport, CagI may have hydrolytic enzymes activity and ATP/GTP enzymes activity.