畜牧兽医学报2025，Vol.56Issue(10)：5104-5114,11.DOI:10.11843/j.issn.0366-6964.2025.10.030
布鲁氏菌分泌蛋白BPE005缺失株的构建及其对GPR126/ADGRG6蛋白的影响
Construction of Brucella Secretory Protein BPE005 Deletion Strain and Its Effect on GPR126/ADGRG6 Protein
摘要
Abstract
In order to explore the mechanism of abortion caused by Brucella secreted protein,a Brucella BPE005 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 S2308△BPE005 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 S2308△BPE005,and the BPE005 backfill strain S2308▲BPE005 was successfully constructed.Every 4 hours,measure the OD600 nm of strains S2308,S2308 △BPE005,and S2308 ▲BPE005,and plot the growth curves of each strain based on time and OD600nm.The multiplicity of infection(MOI)was 100,and the strains S2308,S2308△BPE005 and S2308▲BPE005 were used to infect trophoblast cells HTR-8,and the intra-cellular 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 experi-ment,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 Brucella secretory protein was reduced from 12 h to 44 h,and the growth activity was sig-nificantly 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 S2308△BPE005 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 de-crease.Compared with the S2308 strain,the expression of GPR126 protein in the S2308△BPE005 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 not-ed between the S2308▲BPE005 and S2308 strains..In this study,the stable genetic Brucellase-creted protein BPE005 deletion strain and the supplementary strain were successfully construc-ted,the Brucella secreted protein BPE005 could promote the reproduction of Brucella and pro-mote the intracellular proliferation of Brucella,and the BPE005 deletion strain inhibited the ex-pression 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 Brucella secreted protein on the interaction of GPR126 protein and the pathogenic mech-anism of Brucellain trophoblast cells.关键词
布鲁氏菌分泌蛋白/GPR126表达/基因缺失Key words
Brucella secretory protein/GPR126 expression/gene deletion分类
医药卫生引用本文复制引用
吴婷婷,邓兴梅,关飞虎,郭嘉,张璐,朱德馨,孙志华,曹树珠,徐艺玫,张辉..布鲁氏菌分泌蛋白BPE005缺失株的构建及其对GPR126/ADGRG6蛋白的影响[J].畜牧兽医学报,2025,56(10):5104-5114,11.基金项目
2023年石河子大学高层次科研启动项目(RCZK202358) （RCZK202358）
国家自然科学基金项目(32372973) （32372973）
新疆维吾尔自治区"天池英才"引进计划 （）
