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

doi:10.11843/j.issn.0366-6964.2025.10.030

摘要/Abstract

摘要：

布鲁氏菌T4SS分泌蛋白参与布鲁氏菌引起的母畜流产及胎盘炎症反应，为探究布鲁氏菌分泌蛋白致流产机制，本研究构建布鲁氏菌BPE005缺失株，初步探究分泌蛋白BPE005对胚胎发育关键蛋白GPR126的影响。本试验以Kan基因替换目的基因的同源重组方法，构建BPE005缺失株S2308ΔBPE005，连续培养15代，检测其遗传稳定性；同时构建并电转pBBR1MCS-4-BPE005质粒至BPE005缺失株S2308ΔBPE005中，成功构建BPE005回补株S2308▲BPE005。每4 h检测S2308、S2308△BPE005、S2308▲BPE005菌株的OD_{600 nm}，并根据时间和OD_{600 nm}绘制各菌株的生长曲线。以感染复数(MOI)100，建立S2308、S2308ΔBPE005、S2308▲BPE005菌株侵染滋养层细胞HTR-8模型，通过平板计数方法检测3株菌株胞内生存情况；分别收集侵染后0、4、8、12 h滋养层细胞中的蛋白样和RNA样，通过荧光定量PCR和蛋白免疫印迹方法检测GPR126蛋白表达量。结果显示：本试验成功构建布鲁氏菌分泌蛋白BPE005缺失株和回补株，并且能稳定遗传15代。生长曲线结果显示，与S2308相比，缺失布鲁氏菌分泌蛋白BPE005菌株在12~44 h生长活力都降低，其中，24~44 h平台期时均显著降低(P < 0.001)，但回补株与野生株无显著差异(P>0.05)；胞内生存试验结果表明，与S2308相比，S2308ΔBPE005在HTR-8细胞内存活数量下降，在侵染后12和24 h时显著下降(P < 0.001)。实时荧光定量和蛋白免疫印迹结果表明，与对照组相比，S2308菌株在侵染4 h后，会引起GPR126蛋白表达量的显著升高(P < 0.001)，随后开始下降；与S2308菌株相比，S2308ΔBPE005菌株在侵染后4 h时，GPR126蛋白表达量显著降低(P < 0.001)，而8 h时，GPR126蛋白的显著升高(P < 0.001)，S2308▲BPE005与S2308无显著差异。本试验成功构建稳定遗传的布鲁氏菌分泌蛋白BPE005缺失株和回补株，布鲁氏菌分泌蛋白BPE005能促进布鲁氏菌繁殖，促进布鲁氏菌胞内增殖；BPE005缺失株感染初期抑制GPR126蛋白表达，但中期能促进GPR126的表达。本研究结果为布鲁氏菌分泌蛋白对GPR126蛋白相互作用的影响，以及布鲁氏菌在滋养层细胞中致病机制奠定基础和提供思路。

关键词: 布鲁氏菌分泌蛋白, GPR126表达, 基因缺失

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

中图分类号:

R378.5

吴婷婷, 关飞虎, 郭嘉, 张璐, 朱德馨, 孙志华, 曹树珠, 徐艺玫, 张辉, 邓兴梅. 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.

WU Tingting, GUAN Feihu, GUO Jia, ZHANG Lu, ZHU Dexin, SUN Zhihua, CAO Shuzhu, XU Yimei, ZHANG Hui, DENG Xingmei. Construction of Brucella Secretory Protein BPE005 Deletion Strain and Its Effect on GPR126/ADGRG6 Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5104-5114.

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基因 Genes	引物序列(5′→3′) Primer sequences	产物大小/bp Pruduct size	退火温度/℃ Annealing temperature
BPE005-N (上游同源臂)	F: 5′-CCGGAAATCAACCCGAAAT-3′ R: 5′GACATTCATCCCAGGTGGCTCTTCTCAATCCAGATCGAAAGTCGC-3′	455	56
BPE005-C (下游同源臂)	F: 5′-TCTGGGGTTCGAAATGACCGGAGTTGTCAATACACCTGCATCGG-3′ R: 5′-AATGGCATAGTGGACCTTCTCA-3′	583	56
Kan	F: 5′ GCCACCTGGGATGAATGTC-3′ R: 5′ CGGTCATTTCGAACCCCAGA-3′	1 097	60
BPE005	F: 5′-GGCGCTAGACGACGATATTC-3′ R: 5′-CCTGCTCGATCTGGCCGATA-3′	462	60

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