布鲁氏菌OMP22蛋白的原核表达、纯化及其对巨噬细胞炎性反应因子mRNA表达的影响观察
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摘要
目的原核表达布氏杆菌OMP22蛋白并观察其对巨噬细胞炎性因子mRNA表达的影响。方法在GenBank中查找牛型布鲁氏菌Omp22基因序列并进行引物设计,以其疫苗的全基因组DNA为模板PCR扩增Omp22基因,扩增产物克隆入PEASY-T3载体后测序,测序正确的基因片段与载体pET-32a(+)连接,构建重组表达质粒pET-32a-Omp22,转化感受态细胞BL21(DE3)后,通过IPTG诱导表达融合蛋白OMP22。用胶回收纯化的目的蛋白作用于小鼠巨噬细胞RAW264.7并检测其相关炎性因子mRNA水平。结果结果表明成功构建了布鲁氏菌Omp22蛋白的表达载体,转化DE3后经IPTG诱导表达约35×10~3的目的蛋白,与预期大小相符。Western blot检测该蛋白能被His抗体识别。用纯化的OMP22刺激小鼠巨噬细胞,其IL-1β、IL-6、IL-8、TNF-α等炎性因子表达水平显著降低。结论 OMP22蛋白能降低巨噬细胞的炎性反应表达,为布病的进一步研究提供了理论依据。
Objectives To express the Brucella OMP22 protein in prokaryotic cells and top observe its effect on the expression of macrophage inflammatory factors. Methods GenBank was searched for the gene sequence of Brucella bovis OMP22, and a primer was designed. Genomic DNA of the vaccine strain was used as a template to amplify the OMP22 gene. The product of amplification was cloned into a PEASY-T3 vector and sequenced. The gene fragment was ligated into a pET-32 a(+) vector, and the recombinant expression plasmid pET-32 a-OMP22 was constructed. The plasmid was transformed into BL21(DE3) competent cells. Expression of the fusion protein OMP22 was induced with IPTG, and murine macrophages were treated with the purified target protein. RAW264.7 phagocytes were tested for their level of expression of mRNA of related inflammatory factors. Results An expression vector for Brucella OMP22 protein was successfully constructed. After a plasmid was transformed into BL21(DE3) cells, expression of about 35×103 of the target protein was induced with IPTG. Results were consistent with expectations. The protein was specifically bound by His-OMP22 antibodies according to Western blotting. Mouse macrophages were stimulated with purified OMP22 protein to decrease the levels of expression of inflammatory factors such as IL-1β, IL-6, IL-8, and TNF-α. Conclusion OMP22 protein reduced the expression of an inflammatory response in macrophages. This finding provides a theoretical basis for further research on brucellosis.
Objectives To express the Brucella OMP22 protein in prokaryotic cells and top observe its effect on the expression of macrophage inflammatory factors. Methods GenBank was searched for the gene sequence of Brucella bovis OMP22, and a primer was designed. Genomic DNA of the vaccine strain was used as a template to amplify the OMP22 gene. The product of amplification was cloned into a PEASY-T3 vector and sequenced. The gene fragment was ligated into a pET-32 a(+) vector, and the recombinant expression plasmid pET-32 a-OMP22 was constructed. The plasmid was transformed into BL21(DE3) competent cells. Expression of the fusion protein OMP22 was induced with IPTG, and murine macrophages were treated with the purified target protein. RAW264.7 phagocytes were tested for their level of expression of mRNA of related inflammatory factors. Results An expression vector for Brucella OMP22 protein was successfully constructed. After a plasmid was transformed into BL21(DE3) cells, expression of about 35×103 of the target protein was induced with IPTG. Results were consistent with expectations. The protein was specifically bound by His-OMP22 antibodies according to Western blotting. Mouse macrophages were stimulated with purified OMP22 protein to decrease the levels of expression of inflammatory factors such as IL-1β, IL-6, IL-8, and TNF-α. Conclusion OMP22 protein reduced the expression of an inflammatory response in macrophages. This finding provides a theoretical basis for further research on brucellosis.
引文
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[12] 赵忠鹏. 布鲁氏菌毒力、免疫相关分子的筛选研究[D]. 中国人民解放军军事医学科学院, 2008.
[13] Pappas G, Papadimitriou P, Akritidis N, et al. The new global map of human brucellosis [J]. Lancet Infect Dis, 2006, 6(2): 91.
[14] 殷文武, 孙辉. 中国布鲁氏菌病疫情形势及对策建议[J]. 疾病监测, 2009, 24(7): 475-7.
[15] 廖新华, 苟永慧, 程江. 2013-2015年新疆地区职业暴露高危人群布鲁氏菌病血清学检测结果分析[J]. 中国病原生物学杂志, 2017, 12(5): 444-6.
[16] Moreno E. Retrospective and prospective perspectives on zoonotic brucellosis [J]. Front Microbiol, 2014, 5(2): 213.
[17] Pei J, Ding X, Fan Y, et al. Toll-like receptors are critical for clearance of Brucella and play different roles in development of adaptive immunity following aerosol challenge in mice [J]. Front Cell Infect Microb, 2012, 2(10): 115.
[18] Cloeckaert A, Vizcaino N, Paquet JY, et al. Major outer membrane proteins of Brucella spp.: past, present and future [J]. Veter Microbiol, 2002, 90(1-4): 229-47.
[2] Chen S, Zhang H, Liu X, et al. Increasing threat of brucellosis to low-risk persons in Urban settings, China [J]. Emerg Infect Dis, 2014, 20(1): 126.
[3] 李向阳, 霍晓伟, 武迎红, 等. 布鲁氏菌毒力缺失疫苗株的构建[J]. 基因组学与应用生物学, 2017, 36(1): 273-6.
[4] Araj GF. Human Brucellosis and Its Complications [M]. Neurobrucellosis. Springer. 2016: 7-12.
[5] 崔步云. 中国布鲁氏菌病疫情监测与控制[J]. 疾病监测, 2007, 22(10): 649-51.
[6] Lai S, Zhou H, Xiong W, et al. Changing epidemiology of human brucellosis, China, 1955-2014 [J]. Emerg Infect Dis, 2017, 23(2): 184.
[7] 高彦辉, 赵丽军, 孙殿军, 等. 布鲁氏菌病防治基础研究现状与展望[J]. 中国科学: 生命科学, 2014, 44(6): 628-35.
[8] Ghasemi A, Jeddi-tehrani M, Mautner J, et al. Simultaneous immunization of mice with Omp31 and TF provides protection against Brucella melitensis infection[J]. Vaccine, 2015, 33(42): 5532-8.
[9] Mohammadi E, GolchiN M. Detection of Brucella abortus by immunofluorescence assay using anti-16-kDa outer membrane protein (OMP16) antibody[J]. Comparat Clin Pathol, 2017, 26(6): 1299-304.
[10] 何作萍, 罗佩芳, 胡飞环, 等. 羊布鲁杆菌外膜蛋白19(OMP19)的表达及鉴定[J]. 细胞与分子免疫学杂志, 2016, 32(4): 470-3, 8.
[11] 孙涛, 赵宝, 冉红志, 等. 布鲁氏菌病病原学研究进展[J]. 家畜生态学报, 2014, 35(1): 85-7.
[12] 赵忠鹏. 布鲁氏菌毒力、免疫相关分子的筛选研究[D]. 中国人民解放军军事医学科学院, 2008.
[13] Pappas G, Papadimitriou P, Akritidis N, et al. The new global map of human brucellosis [J]. Lancet Infect Dis, 2006, 6(2): 91.
[14] 殷文武, 孙辉. 中国布鲁氏菌病疫情形势及对策建议[J]. 疾病监测, 2009, 24(7): 475-7.
[15] 廖新华, 苟永慧, 程江. 2013-2015年新疆地区职业暴露高危人群布鲁氏菌病血清学检测结果分析[J]. 中国病原生物学杂志, 2017, 12(5): 444-6.
[16] Moreno E. Retrospective and prospective perspectives on zoonotic brucellosis [J]. Front Microbiol, 2014, 5(2): 213.
[17] Pei J, Ding X, Fan Y, et al. Toll-like receptors are critical for clearance of Brucella and play different roles in development of adaptive immunity following aerosol challenge in mice [J]. Front Cell Infect Microb, 2012, 2(10): 115.
[18] Cloeckaert A, Vizcaino N, Paquet JY, et al. Major outer membrane proteins of Brucella spp.: past, present and future [J]. Veter Microbiol, 2002, 90(1-4): 229-47.