| Helicobacter pylori(H.pylori)is one of the important pathogens of human stomach diseases.It is a Gram-negative spiral micro-aerobic bacteria that can be colonized on human gastric mucosa for a long time.At present,the worldwide infection rate has reached 50%.It has been confirmed that the occurrence of various gastrointestinal diseases such as chronic gastritis,peptic ulcer,gastric cancer and gastric mucosa-associated lymphoid tissue(MALT)lymphoma is closely related to H.pylori infection.The type I H.pylori strain is highly toxic due to its cag pathogenicity island(cag PAI).Cytotoxin-associated gene A(Cag A),an important virulence factor of H.pylori,is synthesized and transported into host cells by the type IV secretion system encoded by the pathogenicity island,resulting in cell dysfunction.At present,the pathogenicity island is known to consist of 27 genes encoding 27 proteins,but the function and pathogenesis of most genes are not fully understood.In this paper,the cag L gene was used as the research object,and its function was predicted by bioinformatics method.The functions of microbiology,molecular biology and immunology were used to verify its function,which laid a foundation for further study on the function and pathogenic mechanism of cag pathogenicity island.Method:1.Calculate various physical and chemical parameters of Cag L by online server and software,analyze its repeat sequence,hydrophobicity curve,antigenicity curve,phylogenetic tree;compare amino acid sequence,analyze Cag L signal peptide and cleavage site and its transmembrane Spiral and coiled-coil cases;predictive algorithms are used to predict their secondary and tertiary structures and search for Cag L functional sites to analyze their possible functions.2.According to the whole genome sequence of H.pylori published by Gen Bank,the suicide plasmid p Bluescript/△cag L::KMr of the cag L gene deletion strain was ligated and identified by restriction enzyme digestion;H.pylori was collected,suicide plasmid was added,and culture was carried out.h,collect bacteria.After the harvested bacteria were identified by urease and bacterial staining,the extracted bacterial genomic DNA was identified by using wild strain 11637 as a control.After gastric cancer epithelial cells were cultured for 2 to 3 days,wild type and cag L gene deletion strains were co-cultured with GES-1 cells;the cells were lysed and subjected to Western blot analysis.3.A total of 158 cases of gastroscopic tissue,colonoscopy,digestive tract tumor tissue and other pathological tissues of the digestive tract were collected from January 2014 to October 2017 in Jiangsu University Hospital.The immunohistochemical staining experiment was used to determine Cag L.Expressions in various organizations.Result:1.Cag L is a conserved protein;there is no signal peptide in Cag L protein;Cag L protein has three functional sites,which are protein kinase C phosphorylation site,casein kinase II phosphorylation site and myristoylation The site of action.2.After digestion,about 651 bp fragment appeared below the electropherogram and about 666 bp fragment appeared below the electropherogram.The results of PCR identification showed that the wild strain obtained a fragment of about 2 kb,and the gene deletion strain obtained a fragment of about 2.5 kb in size.Both the cag L gene deletion strain group and the negative control group failed to detect the Cag A protein.3.By pathological examination of the pathologist,the pathological cell membrane and gland of the digestive tract of different lesions were positive.In conclusion:1.The cag L gene sequence was successfully obtained,and homology analysis was carried out,and the secondary and tertiary structural forms and functional sites of Cag L were predicted.2.The suicide plasmid p Bluescript/△cag L::KMr was successfully constructed,and a cag L gene-deficient strain was obtained.It was proved that the deletion of cag L gene could lead to the loss of CagA protein transport function.3.It was confirmed that the Cag L protein is located in the outer membrane part of the cell and expressed in the digestive tract tissue. |
