| In many pathogens such as Escherichia coli,Salmonella,Vibrio cholerae,and Pseudomonas aeruginosa,MarR6,GntR10,and ArsR6 regulates the expression of genes at the post-transcriptional level which play an important role in the virulence regulation of pathogenic bacteria.The mechanism of the Brucella transcription factors MarR6,GntR10 and ArsR6 has not been fully understood.In this study,we discovered3 transcription factors-MarR6,GntR10 and ArsR6 using the bioinformatics,preparation of polyclonal antibodies,chromosome co-immunoprecipitation assays techniques and the function of their corresponding downstream target genes were also studied.Purpose:(1)Construction of MarR6,GntR10 and ArsR6 gene deletion mutants of B.abortus 2308(S2308).(2)Polyclonal antibodies against Brucella MarR6,GntR10 and ArsR6 were prepared and purified(3)Chromosome co-immunoprecipitation technology and ChIP-Seq screen out target genes and study their biological functions.Method:(1)The upstream,downstream homologous arms and kanna gene were amplified by PCR technology respectively.The fusion PCR technology was used to fuse the three genes and connected pMD19-T.The target genes were exchanged by homologous recombination principle in the genome of the parent strain and detect its growth characteristics.Murine macrophages(RAW264.7)were infected with three deletion strains,and their intracellular survival and inflammatory cytokines were detected.(2)Bioinformatics methods were used to select appropriate fragments of the target gene,and the target fragment was amplified by PCR.Methods The MarR6,GntR10 and ArsR6 recombinant protein of the S2308 strain of Brucellu was amplified using PCR.The fragment was ligated into the vector plas-mid pGEX-4t-1(+)and transformed into DH5 a cells.The recombinant plasmid pGEX-4t-1(+)-MarR6,GntR10 and ArsR6 was constructedand transformed into the Rosetta(DE3)strain.the protein was identified by SDS-PAGE,and the purified recombinant protein was immunized subcutaneously by multi-point injection to test the Japanese white rabbits to determine the serum titer.Affinity chromatography was used for antibody purification,LOWRY assay for antibody protein content,indirect ELISA assay for titer,and Western-blot for antibody specificity.(3)Bacteria were treated with formaldehyde,sonicated,and then chromosome-immunoprecipitated(Chip)technology was used to obtain an enriched target protein-DNA complex that was cross-linked and purified and enriched for DNA fragment PCR analysis.After analysis by Ch IP-Seq technology,the RNAs of S2308,MarR6,GntR10 and ArsR6 deletion strains were extracted and screened for low expression of target genes by RT-PCR;homologous recombination was used to swap the target genes in the parental genome to detect their growth characteristics.,Infected macrophages(RAW264.7)with the deletion strain and detected the intracellular survival and cytokine secretion of the deleted strain BAB_RS21490.Results: The target genes for the regulation of Brucella transcriptional regulators MarR6,GntR10 and ArsR6 were 123,89 and 83,respectively.The BAB_RS21490 deletion strain was successfully constructed and stable hereditary for at least 15 th generation did not recover.The strains had reached the logarithmic growth phase at 14 h of growth,and were basically in line with the growth trend of the parent strain S2308 at 22 h,and intracellular CFU counts.Result:(1)The results showed that the deleted strains MarR6,GntR10 and ArsR6 were successfully constructed for at least 15 generations.The growth tendency was at logarithmic growth phase at 14 h and plateau phase at 24 h and no significant difference with the parental strain.In the experiment of intracellular survival,the ArsR6 and MarR6 deletion strains were significantly lower than the parent strains at 12 h after infecting the cells,and the GntR10 deletion strains were significantly lower than the parent strains at 4 h and 12 h after infecting the cells.The secretion levels of TNF-α and IL-1β in the four infection groups were higher than those in the PBS control group at 0 h,4 h,12 h,and 24 h in the three deletion strains and parental strains.The infections in the ΔGntR10 group were 4 h after infection.The secretion of TNF-α was significantly higher than that of the parental strain at 24 h,but there was nosignificant difference between ΔMarR6 and ΔArsR6 and the parent strain.(2)Using bioinformatics analysis,the MarR6,GntR10 and ArsR6 genes were selected to be 255 bp,300 bp and 228 bp,respectively.The position of PCR amplification was consistent with the target band.SDS-PAGE after self-induced expression and purification showed MarR6.The GntR10 and ArsR6 fusion proteins were at 34 KD,37 KD and 32 KD,respectively;the titer could reach 1:512000,and the hybridization band was detected by Western-Blot;the protein content was 3 mg/L,2 mg/L and 2.54 respectively.mg/L.(3)The BAB_RS21490deletion strain was significantly lower than the parent strain S2308 at 4 h and 12 h after infection.The expression level of IL-1β was significantly lower in the infected strains than in the parental strain S2308 at4 h and 12 h after infection,and significantly higher than the parental strain at 24 h;the expression level of TNF-α was 0 h and 24 h after infection in the deleted strain.Significantly higher than the parent strain 2308,12 h was significantly higher than the parent strain S2308.Conclusion:(1)The MarR6,GntR10 and ArsR6 deletion mutants of Brucella were successfully constructed and verified,and they were stable for at least 15 passages.The growth curve showed a good growth trend and there was no significant difference in the parental strain;the deletion strain Brucella was in the bacterial cell The in vivo viability weakened;the secretion of TNF-α and IL-1β increased after infection with the GntR10-deleted strain,which laid a solid foundation for the study of transcription factor function of S2308.(2)The recombinant proteins Mar R6,GntR10 and ArsR6 of S2308 were successfully cloned and expressed.The expressed recombinant protein was immunogenic with a titer of 1:512000.A high-purity,high-titer polyclonal antibody was prepared.The establishment of a sedimentation foundation.(3)295 target genes of MarR6,GntR10 and Ars R6 were screened;among them,the GntR10 target gene BAB_RS21490 was lower than S2308.BAB_RS21490 promotes the intracellular survival of Brucella and inhibits the secretion of inflammatory cytokines IL-1β and TNF-α.Finally,this study successfully constructed and identified the stability of Brucella MarR6,GntR10 and ArsR6 mutants after serial transfer at least 15 generations.There was no difference in extracellular survival ability between the parental strains and these mutants.However,the intracellular survival rates of the mutants were much lower than parental strain.The recombinant of MarR6,GntR10,ArsR6 of S2308 were cloned and its protein was expressed,and polyclonal antibodies with high purity and titers were also prepared;the number of target genes regulated by of Brucella transcription regulators MarR6,GntR10 and ArsR6 were 123,89 and 83.The Brucella can inhibit the occurrence if inflammation after infected macrophages through up-regulate the gene expression of BAB_RS21490,the production of inflammatory cytokines TNF-α and IL-1β were blocked. |
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