Construction of Brucella Effector Protein BspE Gene Deletion Strain and Its Biological Characteristics
[Objective]The purpose of this study was to construct the BspE gene deletion strain of Brucella abortus(B.abortus)A19 strain,explore the growth characteristics of survival,adhesion and invasion in host cells,and also observe the subcellular localization of BspE protein of B.abortus A19 during infection.[Method]B.abortus A19 was used as the research object.The Brucella effector protein BspE gene deletion strain A19△BspE was constructed by homologous recombination and SacB reverse screening technology,and its complement strain A19C△BspE was constructed by plasmid complementation method.The growth of three strains was measured and the influence of BspE gene deletion on the survival,adhesion and invasion of Brucella in cells was analyzed.The localization of BspE protein in RAW264.7 cells was observed by indirect immunofluorescence assay.[Result]PCR results of the bacterial solution showed that a specific band with a size of 2 060 bp was obtained,and the BspE gene deletion strain A19△BspE was successfully constructed.Western blotting results showed that a BspE-flag band with a size of approximately 14.59 ku was obtained,and the complementation strain A19C△BspE was successfully constructed.The growth curve results showed that under the same culture conditions,there was no significant difference among B.abortus A19,A19△BspE and A19C△BspE(P＞0.05),all of them reached the logarithmic growth phase at 8 h and entered the platform phase at 32 h.The results of intracellular proliferation test showed that the viability was not significantly different from B.abortus A19,A19△BspE and A19C△BspE during 48 h post infection in RAW264.7 cells(P＞0.05).The results of bacterial adhesion and invasion test showed that the ability of A19△BspE to adhere and invade RAW264.7 cells was not significantly different from that of B.abortus A19 and A19C△BspE(P＞0.05).The result of indirect immunofluorescence assay showed that BspE protein was mainly distributed in the perinuclear of RAW264.7.[Conclusion]This study proved that the deletion of BspE gene did not affect the growth of Brucella in vitro and the survival,adhesion and invasion in RAW264.7 cells,and BspE protein was mainly located in the perinuclear region,which laid a foundation for the biological function and pathogenic mechanism of Brucella effector protein BspE.
Brucellaeffector protein BspEmutant strainbiological characteristics
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维普
