| Campylobacter jejuni is pathogen majorly caused food-borne diarrhea worldwide.It has been found that the pathogenesis of campylobacter is closely related to the formation,adhesion,invasion and toxin of flagella on the host epithelial cells.Cj0440 c is a putative transcriptional regulator protein,which was up-regulated in the erythromycin resistant C.jejuni.In the early study,when cj0440 c was knocked out in C.jejuni NCTC11168,the flagellum was broken and the flagella genes were differentially expressed.Since the regulation mechanism of Cj0440 c is not very clear.Studies have shown that,the homologous protein of Cj0440 c is TenA,TenA is a thiaminase II,TenA can take part in the synthesis of thiamine.So we suspected that Cj0440 c protein also has the role of thiaminase II.In order to study the function of cj0440 c,Observed the expression of cj0440 c and flagella related genes under the resistant bacteria of different drugs.To find out the relationship between cj0440 c and flagella related genes.We get Cj0440 c protein by prokaryotic expression.Design enzymatic hydrolysis experiment to verify whether Cj0440 c protein is involved in the metabolic pathway of thiaminase.Construct the over expression vector of C.jejuni,Using chromatin immunoprecipitation(Chip)to look for the target genes which regulated by Cj0440 c.Verified the interaction between Cj0440 c protein and flagellar correlation genes was by Gel Shift Assays(EMSA).Previous studies have found that cj0440 c was up-regulated in erythromycin resistant strain,so detected the MIC and MPC of C.jejuni for nine antibiotics,and determine the mutant selection window(MSW)of every antibiotics.Choose the drug-resistance bacteria in MSW,analyzed the expression of cj0440 c and the flagella related genes.In C.jejuni NCTC11168,cj0440 c was up-expression in all other resistance strains except in tigecycline resistance bacteria,cj0440 c and cj1338 c were up-regulated in erythromycin resistant strain,the result agreed with the result of the expression of cj1338 c in cj0440 c mutant strain.In the selected two kinds of aminoglycoside drug and two kinds of tetracycline drug resistant bacteria,the expression of the other four genes was consistent with cj0440 c.In the two kinds of fluoroquinolone drug resistant bacteria,cj0440 c,cj0043 and cj0697 were up-expression,but cj1338 c and cj1339 c were down-expression.In C.coli ATCC33559,cj0440 c was down-expression in all other resistance strains except in erythromycin,azithromycin and gentamicin resistance bacteria.In erythromycin and gentamicin resistant strain cj0440 c and other flagellin genes were up-regulated.When cj0440 c was down-regulated,at least one gene in the four flagella genes will be downregulated,verified cj0440 c can cause the differential expression of flagella related genes.Using the plasmid pET28 a to constrct recombinant vector P-440 by double enzyme digestion,and using E.coli BL21 to express the protein.To contect the protein by the His tag of the plasmid pET28 a.Using Ni-NTA to purify protein,and find the function of Cj0440 c protein by design enzymatic experiment.Using the plasmid pRY112 to construct the cj0440 c over expression vector which can express in C.jejuni to find the genes which regulated by Cj0440 c in vivo.After constructed the over expression vector,tried transforme the over expression vector into C.jejuni by natural transformation,electrical transformation and joint transfer,but it all ended in failure.So changed the method to find the target genes in vitro.According to the previous study of the lab,choose the genes cj1026 c,cj1729c,cj1466,cj0687 c,cj0697,cj0698,cj1632 c,cj1339c and cj1462 to do electrophoretic mobility shift assays(EMSA).The results did not show that the Cj0440 c protein was able to directly bind gene promoter fragments.In conclusion,In all drug-resistance bacterias,cj0440 c and other flagella related genes all differential expression,and the change trend is consistent with the previous research results.When cj0440 c down-regulated expression,at least one of the four selected flagellated genes will be down-regulated expression.It is indirectly verified that cj0440 c can participate in the synthesis metabolism of flagellar.Cj0440 c protein was successfully expressed in this study,the enzymatic hydrolysis experiment demonstrated that the gene did not have the effect of thiaminese.In electrophoretic mobility shift assays,Cj0440 c can't direct conbine with the promotor of nine genes,maybe Cj0440 c can't regulate these genes.Preliminary judgment,maybe cj0440 c through other genes to regulated the flagella genes.The study of this topic helps to understand the interrelationship between flagellar genes.It has reference significance for improving the regulation mechanism of C.jejuni flagella. |
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