| Part 1 Sequence characteristic of 3'region of cagA in helicobacter pylori strains isolated from patients with different gastroduodenal diseasesObjective:To compare the sequence characteristic of 3'region of cagA in helicobacter pylori(H.Pylori)strains isolated from patients with different gastroduodenal diseases and find out their relations to clinical diseases.Methods.A total of 41 Helicobacter pylori strains was isolated from gastric biopsy samples of 13 patients with chronic superficial gastritis,13 patients with duodenal uncler and 15 patients with gastic cancer.The 3'region of cagA DNA fragments were amplified by PCR from genomic DNA of clinical isolated strains.The DNA fragments were sequenced by automatic sequencer(ABI3730),and the corresponding amino acid sequences were deduced by Transeq program from EBI(European Bioinformatics Institute).Sequence alignment and similarity analysis was performed with the Vector NTI advance 10,then analyse Sequence characterisitics and their correlation with the gastroduodenal diseases.Result:(1)36(87.89%)of the 41 clinical isolated H.Pylori strains were cagA gene positive.The positive rates of cagA in DU,GC were both 100%,significantly higher than in CSG(61.5%,P＜0.05).(2)The amplified PCR products of cagA 3' region from thirty-six clinical isolated strains ranged from 615～664,there are a great quantity insertion,deletion,substitution,repetition of basic group,amino acid in the nucleotide and deduced amino acid sequences and showed a low level of similarity.Among the nucleotide and deduced amino acid sequences of the 3'region of cagA from 36 clinical isolated H.Pylori strains,the identity position was 19.7%,36.3%respectively.Based on having different SHP-2 binding sequence,the amino acid sequences of the 3'region of cagA could be classified into two types:East Asian type(69.4%)and western type(30.6%).The number of EPIYA motif was three in all deduced amino acid sequences,and three patterns of amino acid segments were observed immediately downstream of the EPIYA motif,named as R2,R3,R4 respectively.The East type and westem type have different R3,R4 respectively.There were a high level of similarity among the nucleotide,amino acid sequences of cagA 3'region from both East Asian type and westem type,the identity position of basic group,amino acid from East Asian type cagA was 30.7%,59.6%,corresponding from west type was 38.6%,73.1%.The prevalence of East Asian cagA positive strains was significantly higher in patients with duodenal ulcer(92.31%) and gastric cancer(80%)than in patients with chronic superficial gastritis (12.5%,P＜0.05).(3)There were a high level of similarity among the nucleotide,amino acid sequences of 3'region of cagA from each gastroduodenal disease,the identity position of basic group,amino acid from CSG group was 70.7%,54%,corresponding from DU group was 30.8%,48.4%,GC group was 22.3%,43.2%;The similarity difference had no statistical significance(P＞0.05).Conelusion:(1)The positive rate of cagA high,the cagA positive stains were related to DU,GC more closely.(2),The nucleotide and deduced amino acid sequences of 3'region of cagA have polymorphism,the diversity was caused by insertion,deletion,substitution,repetition of basic group,amino acid.(3)The 3'region of cagA gene could be classified into two types:EastAsian type and western type,both type showed a high level of similarity.(4)There were a high level of similarity among sequences of 3'region of cagA from each gastroduodenal disease,the East Asian cagA-positive strains were related to GC,DU more closely.Part 2 Polymorphism of 3'region of cagA in helicobacter pylori strains and its clinical significanceObjective:To explore the Polymorphism of 3'region of cagA in helicobacter pylori strains and its relevances with regions and clinical diseases.Methods:Searched out 95 items of cagA gene complete sequence and 387 items of nucleotide and corresponding amino acid sequence of 3'region of cagA gene of helicobacter pylori(H.Pylori)from NCBI database on network,there still were 36 items of 3'region of cagA from 41 clinical isolated H.Pylori strains(13 patients with chronic superficial gastritis,13 duodenal uncler and 15 patients with gastic cancer);Vector NTI advance 10 software was used to multiple-sequence alignment and similarity analysis of the 36 items from clinical isolated strain and the 256 items of 3'region of cagA coming from network and of which the background of disease had been known.The softwares- Clustal X(version 3.5),Phylip(version 3.5),Treeview(version 1.61)-were used in phylogenesis analysis of 91 items of cagA gene complete sequence;then aggregate analyse of the Polymorphism of 3'region of cagA and its relevances with regions and clinical disease.Result:(1)The phylogenetic tree contructed on the basis of the full-length CagA sequences showed that the cluster relationship of strains from Asian were close,and the cluster relationship of strains from west were close.(2)The comparison of complete amino acid sequence of cagA showed that the 5'region of cagA gene was relatively conservative,and the differences among the 3'regions were obvious;the identity position of amino acid among complete amino acid sequences of cagA is 27%,the similarity is relatively low,besides lots of insertion,deletion,substitution,there were many repeated sequences in 3'region.(3)There were 6 kinds of repeated sequences in amino acid sequences of 3'region,named as C1,C2,C3,C4,C5,C6;the sequences-C3, C4,C6 could be classified into two types based on different amino acid composition:East Asian type(Cneast)and western type(Cnwest).The basic structure of amino acid sequences of 3'region was C1-C2-C1-C3east/C3west -C4east/Cwest-C5-C1-C6easr/C6west.(4)A few amino acid sequences of 3'region of cagA were longer,they had different number of repeated sequences and different combination types,based on this,these few amino acid sequences of 3'region of cagA were divided into 10 types,the types of 1,7,9,10 only could be found in East Asian strains,on the contrary,the types of 2,3,5,8 only could be found in west strains, types of 4,6 could be found both in East Asian and west strains.(5)The detection rate of strains with more than 4 EPIYA motif in atrophic gastritis group was 11.90%,and in peptic ulcer group was 1.09%,the difference between them had statistical significance(P＜0.05),87.5% strains with more than 4 EPIYA motif were detected in atrophic gastritis and gastic cancer group.The detection rate of strains with multiple EPIYA-C/D site in gastic cancer group(13.04%)was obviously higher than in peptic ulcer group(4.35%,P＜0.05).In the sequences of which C4 repeated 3 times,the comparision between GC(10.14%)and PU (1.09%)showed statistical significance(P＜0.05).The number of other repeated sequences in different gastroduodenal diseases had no statistical significance(P＞0.05).The detection rate of the sixth type strain in gastic cancer group(11.59%)was obviously higher than that in peptic ulcer group(2.17%,P＜0.05).Conelusion:(1)Polymorphism of variable region of 3'region of cagA gene in H.Pylori strains was significant,the differences were mainly caused by the type,number and combination type of repeated sequences.(2) CagA+H.pylori with more than 4 EPIYA motif maybe was correlated with atrophic gastritis and gastic cancer.(3)CagA+H.pylori with multiple EPIYA-C/D site and the sixth type maybe was correlated with gastic cancer. |
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