Font Size: a A A

Cloning And Expression CagM Gene Of Helicobacter Pylori And Bioinformatics Analysis

Posted on:2011-02-28Degree:MasterType:Thesis
Country:ChinaCandidate:S W TianFull Text:PDF
GTID:2120360302493726Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Objective:Clone cagM(HP0537) full-length gene from Helicobacter pylori(Helicobacter pylori,H.pylori) NCTC 11637,and construct its prokaryotic expression vector.To express and purify CagM protein,to analyze its antigenicity and produce antibodies,use bioinformatics method to analysis its physical and chemical characteristics,and predict its structure and function,in order to further study the role of CagM protein in typeⅣsecretion system of Helicobacter pylori and the Pathogenic mechanism of Helicobacter pylori.Methods:Oligonucleotide primers were designed according the sequence of H.pylori 26695 in GenBank,using Primer Primier 5.0 software. And in order to facilitate cloning and ensure the exactness of insert direction,we designedly joined some inscribed enzyme points into primers. H.pylori cagM gene was amplified from the genome DNA of H.pylori NCTC 11637 by PCR.The purified.PCR products were cloned into pGEM-T vector with T-A Cloning and transformed into competent E.coli DH5α.It was detected by double restriction enzyme digestion.The nucleotide sequence of the insert was determined by Sangon.Sequencing results will be submitted to GenBank,access to gene accession number. Nucleotide sequences and amino sequences encoded by cagM gene was analyzed with the sequence alignment,as well as its amino acid composition,molecular weight,isoelectric point,hydrophilicity, hydrophobicity,antigenicity and other physical and chemical properties was analyzed using bioinformatics.And then inserted CagM fragment directionally into pET-28a(+) vector to construct pET-28a(+)-cagM expression vector and transformed it into expression host strain E.coli BL21,by kanamycin resistance and double-enzyme digestion to identify positive clones.Then IPTG to induce its expression,and to optimize the expression conditions(induction temperature,induction time,and IPTG concentration),finally purified the CagM proteins with Ni2 +-NTA resin. Prepared antibody of the protein by immunizing rabbit,and detected the titer by using ELISA assay.With biological databases and bioinformatics software to analyze CagM protein signal peptide,transmembrane domains, sub-cellular localization and protein family,as well as to predict their secondary structure,tertiary structure and protein functional sites.Results:1.The cagM gene of H.pylori NCTC 11637 is 1131 base pairs long, which encodes a product of 376 amino acids.GenBank accession number is GU269568.The sequence analysis for cagM showed that it shares 96%~99%homology with other strains of H.pylori in Genbank and Amino acid homology is 98%~99%.2.With software analysis found that,CagM protein contains 65 basic amino acids,52 acidic amino acids,126 hydrophobic amino acids,and 111 non-polar amino acids,molecular weight is about 43.76199kD,isoelectric point is 9.3,Davis,Botstein,Roth melting point is 78.56℃.CagM protein has multiple hydrophobic regions and multiple hydrophilic regions,where hydrophilic areas are mainly distributed in the N terminal.CagM have strong antigenicity.3.Successfully constructed pET-28a(+)-cagM prokaryotic expression vector and optimized of expression conditions.The best expression conditions is IPTG final concentration of 1.0mmol/L,temperature 30℃, induction time of 4h.The relative molecular mass(Mr) of recombinant protein CagM is about 43.7kDa after Ni2+-NTA purified,in line with the forecast results.CagM recombinant protein immunized of rabbits to obtain anti-CagM polyclonal antibody,the titer achieved 1:1.6×105. 4.Bioinformatics analysis found that,20 amino acids of CagM protein N-terminal is the signal peptide,splice site at 21 amino acids,sub-cellular located in the bacterial periplasmic space,the secondary structure have more than 60%α-helix,about 10%of random coil,small part ofβ-folding and turns.Its tertiary structure likes a saddle-like,the N-terminal and C-terminal is globular part and the middle part is of the single-stranded form.5.CagM belong to the 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 typeⅣsecretion system,in the Bacterial Periplasmic space.Conclusion:1.Successfully cloned cagM gene,which is one of the important genes of TFSS encoded by cag PAI in H.pylori.2.We have constructed the prokaryotic expression vector,obtained CagM recombinant protein and prepared polyclonal antibody.3.We obtained the majority of physical and chemical properties feature data of CagM protein,and predicted its high-level structure.4.We resolved CagM protein's sub-cellular localization,knew its own protein family,and analyzed its functional sites.
Keywords/Search Tags:Helicobacter pylori, cagM, prokaryotic expression, structure prediction, functional analysis
PDF Full Text Request
Related items