| Brucellosis is an important zoonitic infectious disease worldwide,caused by Brucella spp.,leading to an abortion in females and sterility in males.Human can be infected by infectious animals or eating some food with Brucella.It poses a great threat for the development of animal husbandry and human health.Brucella is a facultative intracellular bacterium,which can invade and replicate within host cells,then establish chronic infection in host.But the pathogenesis of Brucella has not be elucidated.In this study,in order to explain a novel pathogenesis of Brucella,the functional research of novel virulence genes vhpA and vhpB1/2 was performed,and a plasmid for promoter activity determination of Brucella genes was constructed,which may benefit investigation of Brucella virulence genes.1.Functional research of Small protein VhpA in pathogenic process of BrucellaThe vhpA gene encodes for a highly conserved small protein in Rhizobiales spp.based on bioinformatics analysis.To investigate the role of this gene,a deletion mutant and complement strain were constructed.The phenotype analysis of vhpA mutant showed that the vhpA mutant was constructed successfully without phenotype changes.Virulence testing showed that vhpA gene is necessary for Brucella virulence.The results of a cell infection experiment showed that vhpA gene was not associated with Brucella adherence to and invasion of HeLa cells,and intracellular survival within RAW264.7 cells.The results of sensitivity testing showed that the vhpA mutant had similar sensitivity to hydrogen peroxide,polymyxin B,and sodium nitroprusside as the wildtype strain.Additionally,further research showed that the homologous vhpA genes of Sinorhizobium meliloti and Agrobacterium tumefaciens could not restore virulence of the vhpA mutant.2.Regulatory mechanism research of small protein VhpA for Brucella virulenceIn order to investigate the function of vhpA gene,RNA-seq analysis showed that deletion of the vhpA gene affected the expression patterns of multiple Brucella genes,and the main four up-regulated genes and five down-regulated genes were further confirmed using quantitative real-time PCR analysis.Subsequently,a series of over-expression strains were constructed,and virulence testing showed that over-expression vhpB1 and vhpB2 genes significantly reduced virulence of the wild-type strain.VhpB1 and VhpB2 proteins are highly homologous based on bioinformatics analysis.The PCR result showed that vhpB1 and vhpB2 genes can transcribe to a polycistronic mRNA.The vhpB1/2 mutant was constructed,the virulence testing showed that deletion of vhpB1/2 significantly reduced virulence of the WT strain.vhpB1 and vhpB2 genes were annotated as hypothetical genes with unknown function,and we found that they can encoding two small proteins in Brucella by western blot,promoter activity analisis and over-expression of ORF within WT strain.3.Construction and application of a novel plasmid for promoter activity determination of Brucella genesvhpB1/2 gene may precisely regulate Brucella virulence through different levels of transcription and expression.Promoter as an important structure to initiate the transcription and expression.The transcription and expression of vhpB1/2 gene under special conditions can be determined by promoter activity.Its precise regulation mechanism can be analyzed by promoter activity of vhpB1/2 gene.Based on the highly sensitive of NanoLuc luciferase,the purified recombinant NanoLuc luciferase protein and polyclonal antibody was prepared in this study.And the plasmid for promoter activation determination of Brucella genes was constructed based on NanoLuc luciferase.And the feasibility was verified by the determination of bcsp31 and virB promoter activity. |
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