| Background and objective:The membrane protein and the H.pylori's planting, gastric mucosa damage,inflammatory mediators secretion and neutrophils infiltrating the etc have closerelations, at the same time it in the main ingredients stimulate an immune response toinduce strong strain specific immune responses, this in the cause of H.pylori plays animportant role. H.pylori infection of the outcome after mainly by bacterial factors,host factors and environmental factors of the three factors, especially bacterial factorsin the outcome of the H.pylori infection factors play an important role in diversity,H.pylori virulence factors is an important reason for genetic polymorphism too.Former H.pylori virulence stduy focus on urea enzyme, cytotoxin-associatedprotein(Cag), vacuolating cytotoxin A (VacA) etc, and years of study found that theouter membrane protein and H.pylori colonize inflammatory mediators, the secretionof gastric mucosa, damage and neutrophils infiltrating the has close relation. The aimwas to construct a recombinant plasmid that expresses the H.pylori sialic acid-bindingadhesion(SabA), the recombinant protein was identified as H. pylori outer membraneprotein SabA by Western blot. Analysis from the Jiangxi clinical H.pylori strainsouter inflammatory protein A (OipA), blood-group antigenbinding adhesin (BabA),sialic acid-binding adhesin (SabA) gene sequence characteristics and22H.pylorisequences from East Asian and non-East Asian region to compare, analyze thecharacteristics of different regions of the sequence. Proven our outer membraneprotein of the genetic characteristics of great significance for the study of itsrelationship with the disease and laid the foundation for the SabA as the targetantigen.Methods:1. Cloning and expressing of H. pylori sialic acid-binding adhesion:(1) The primers were designed according to the sequence of the H. pylori26695sabA gene;(2) PCR products were inserted into expression vector pGEX-4T-1,and transformed into E.coli BL21strain for the expression;(3) The recombinant protein was identified by MALDI-TOF-MS and theantigenicity was tested by Western blot.2. Gene sequence analysis of h. pylori outer membrane protein (OipA, BabA,SabA):(1) Pre-separation of the154clinical H.pylori strains for resuscitation culture,and extraction its genomic DNA;(2)For H.pylori membrane protein (OipA, BabA, SabA) gene design of morespecific full-length sequencing primers;(3) Amplification of purpose gene, and products sent to the company sequenced;(4) Use of bioinformatics software MEGA, AlignX, ClustalX2and relatedinformation database for DNA sequence and aa sequence analysis;(5) Jiangxi clinical isolate sequences and22different regions of the H.pyloristrain sequences were compared, analysis of different regions of the sequencecharacteristics, to identify the sequence specificity of strains of Jiangxi.Results:1. H.pylori sialic acid binding adhesin Cloning and expression of results:(1)Get H.pylori26695sialic acid-binding adhesion gene, and identification ofrecombinant plasmid pGEX-4T-1-sabA by restricted endonuclease enzymes, sabAsuccess inserted into pGEX-4T-1vector.(2)The recombinant plasmid pGEX-4T-1-sabA expressed in E. coli BL21andwas identified by MALDI-TOF-MS, the recombinant protein is H. pylori outermembrane protein SabA, and the protein has antigenicity.2. H.pylori outer membrane protein (OipA, BabA, SabA) Gene SequenceAnalysis of results:(1)Amplified by the outer membrane proteins of clinical strains OipA, BabA,SabA three genes, and multi-primer PCR of three genes positive rate was100%. DNAsequences translated into amino acids, predict the protein OipA protein-positivedetection rate was99.33%, BabA and SabA protein-positive detection rate were75.89%and61.90%. babA gene sequences are52.43%of the base sites of a mutationwas significantly higher than49.28%of sabA gene sequences, translated into amino acid sequences of BabA amino acid sequences are42.38%of the amino acid pointmutations in significantly below the sabA amino acid sequence46.96%, indicating thethe babA base sites of mutation in the majority of synonymous mutations, the DNAsequence changes do not affect the amino acid changes.(2)DNA sequence of the cluster analysis results:①oipA gene257mutation sites, accounting for27.81%of the full-length gene,strains with different parts of the sequence alignment Jiangxi region sequence ofstrains CT repeat number is not obvious. Sequence Clustering can be divided intoseven categories, which come from non-East Asian region strain sequences wereclustered into a subcategory, the sequence of strains from East Asia, respectively, andJiangxi strain sequences were clustered into different subclasses.②babA gene1209mutation sites, accounting for52.43%of the full-lengthgene, The existence of a high mutation region than the entire babA gene genesequence600~1100sites. Sequence Clustering can be roughly divided into twocategories, the strains from non-East Asian region sequences were clustered into acategory, the sequence of strains from South Korea were clustered into a subcategory,the sequence of strains from Japan and Jiangxi strain sequence clustered into differentsubclasses.③sabA gene1021mutation sites, accounting for49.28%of the full-lengthgene, Than found sabA the entire gene sequence0~100,200~1200sites, there are twohigh mutation area, strains with different parts of the sequence alignment Jiangxiclinical isolate of sequence does not exist CT repeat sequences. Clustering can bedivided into two categories, the sequence of strains from East Asia and Jiangxi strainsequences were clustered into different subclasses.(3) amino acid sequence of the cluster analysis results:①oipA amino acid sequence of81amino acids mutated, accounting for26.30%of the long amino acid sequence, its isoelectric point between9.42to10.03,with an average of9.62; instability index between16.12to23.81, with anaveragevalue of18.91; fat index between77.30to82.11, with an average of79.73;hydrophilic value between－0.385to－0.255, with an average of－0.341. Thededuced amino acid sequence clustering analysis, the amino acid sequence is broadly divided into seven categories, and the DNA sequence clustering is the corresponding.②babA amino acid sequence of317amino acids mutated, accounting for42.38%of the long amino acid sequence, its isoelectric point between6.15to11.00,with an average of9.18; instability index between－1.86to43.61, with an average of21.59; fat index between0to92.08, with an average of67.21; hydrophilic valuesbetween－1.575to0.020, with an average of－0.426. The deduced amino acidsequence clustering analysis can be divided into three categories, the No.142strainamino acid sequence and amino acid sequences of other strains have a lot ofdifference.③sabA amino acid sequence of1021point amino acid mutated, accounting for46.96%of the long amino acid sequence, its isoelectric point between4.93to12.31,with an average of9.29, the instability index between7.66to62.90, with an averageof31.37; fat index between71.78to149.41, with an average of90.60; hydrophilicvalues between－0.761to1.200, with an average of－0.181. The deduced amino acidsequence of the cluster analysis, two major categories can be broadly classified.Conclusion:1.The gene sabA of H.pylori26695was successfully cloned and expressed inE.coli BL21, and the protein expressed has good antigenicity.2.H.pylori gene oipA babA and sabA have sequence polymorphism andsequence changes and foreign is not the same, sequence has obvious regionalcharacteristics.3.Sequence found on the clustering of clinical strains sequence withpolymorphism, the sequence of strains of different regions have differentcharacteristics, and our strain sequence with the East Asian region is relatively close,there are great differences with the non-East Asian region. |
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