Abstract:

In order to explore the mechanism of abortion caused by Brucellasecreted protein, a BrucellaBPE005 deletion strain was constructed to preliminarily explore the effect of secreted protein BPE005 on GPR126, a key protein in embryonic development. In this experiment, the BPE005 deletion strain S2308ΔBPE005 was constructed by homologous recombination method of replacing the target gene with Kan gene, and its genetic stability was tested by continuous culture for 15 generations. At the same time, the pBBR1MCS-4-BPE005 plasmid was constructed and electrotransposed into the BPE005 deletion strain S2308ΔBPE005, and the BPE005 backfill strain S2308▲BPE005 was successfully constructed. Every 4 hours, measure the OD_{600 nm} of strains S2308, S2308△BPE005, and S2308▲BPE005, and plot the growth curves of each strain based on time and OD_{600 nm}.The multiplicity of infection (MOI) was 100, and the strains S2308, S2308ΔBPE005 and S2308▲BPE005 were used to infect trophoblast cells HTR-8, and the intracellular survival of the three strains was detected by plate counting. Protein and RNA samples were collected from trophoblast cells at 0, 4, 8, and 12 h after infection, respectively, and the protein expression of GPR126 was detected by real-time PCR and western blot. In this experiment, the deletion strain and the supplemental strain of Brucella secreted protein BPE005 were successfully constructed, and the gene could be stably inherited for 15 generations. The results of the growth curve showed that compared with S2308, the growth viability of the BPE005 strain lacking Brucellasecretory protein was reduced from 12 h to 44 h, and the growth activity was significantly decreased from 24 h to 44 h plateau stage (P < 0.001), but the resupplementation strain reversed this result. The results of intracellular survival assay showed that the survival of S2308ΔBPE005 in HTR-8 cells decreased compared with S2308, and decreased significantly at 12 h and 24 h after infection (P < 0.001). The results of real-time fluorescence quantification and western blotting showed that compared with the control group, the expression of GPR126 protein in S2308 strain increased significantly at 4 h after infection (P < 0.001), and then began to decrease. Compared with the S2308 strain, the expression of GPR126 protein in the S2308ΔBPE005 strain was significantly decreased at 4 h after infection (P < 0.001), while the expression of GPR126 protein was significantly increased at 8 h (P < 0.001). No significant differences are noted between the S2308▲BPE005 and S2308 strains.. In this study, the stable genetic Brucellasecreted protein BPE005 deletion strain and the supplementary strain were successfully constructed, the Brucellasecreted protein BPE005 could promote the reproduction of Brucella and promote the intracellular proliferation of Brucella, and the BPE005 deletion strain inhibited the expression of GPR126 protein in the early stage of infection, but could promote the expression of GPR126 in the middle stage. The results of this study lay a foundation and provide ideas for the effect of Brucellasecreted protein on the interaction of GPR126 protein and the pathogenic mechanism of Brucellain trophoblast cells.

Key words: Brucellasecretory protein, GPR126 expression, gene deletion