牛分枝杆菌PtpA蛋白对NF-κB信号通路的调控机制
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摘要
【目的】研究牛分枝杆菌Mycobacterium bovis PtpA蛋白对免疫应答相关的NF-κB信号通路的影响,以揭示PtpA蛋白在机体免疫应答中的作用。【方法】构建PtpA基因真核表达载体FLAG-PtpA,并将其转染到HEK293T中,进行SDS-PAGE分析及Western blot检测。激活NF-κB信号通路后,通过双荧光素酶试验和qPCR方法探究PtpA蛋白对NF-κB信号通路的影响。【结果】成功构建PtpA基因真核表达载体FLAG-PtpA,转染HEK293T后经SDS-PAGE分析,相对分子质量约为22 000处可见特异性蛋白条带。Western blot结果显示,表达产物可与一抗特异性结合,证明该蛋白是PtpA蛋白。双荧光素酶试验中,转染2～24 h,试验组与对照组萤火虫荧光素酶和海肾荧光素酶的相对荧光强度的比值差异显著(P<0.05);转染2 h后,对照组萤火虫荧光素酶和海肾荧光素酶的相对荧光值是试验组的2.93倍,说明PtpA蛋白对NF-κB信号通路激活早期具有显著的抑制作用。qPCR结果显示,转染2 h后,对照组的IL-6、GM-CSF、BIRC-2和BIRC-3的表达量分别是试验组的3.93、3.42、2.17和2.30倍(P<0.01);转染4 h后,对照组的IL-6、GM-CSF、BIRC-2和BIRC-3的表达量分别是试验组的4.26、3.93、2.36和2.50倍(P<0.01),说明PtpA蛋白对NF-κB信号通路相关的细胞因子(IL-6、GM-CSF、BIRC-2、BIRC-3)在免疫早期具有显著抑制作用。【结论】qPCR结果与双荧光素酶试验结果一致,表明牛分枝杆菌PtpA蛋白对NF-κB信号通路的影响主要发生在早期。本研究为后续研究有效的结核病防控药物提供了理论基础。
【Objective】To investigate the effect of Mycobacterium bovis PtpA protein on NF-κB signaling pathway which is related to immune response, and reveal the role of PtpA protein in the body's response to immunity.【Method】The PtpA gene eukaryotic expression vector FLAG-PtpA was transfected into HEK293T for SDS-PAGE analysis and Western blot detection. After activation of NF-κB signaling pathway, the effects of PtpA protein on NF-κB signaling pathway were investigated by dual luciferase assay and qPCR.【Result】The eukaryotic expression vector FLAG-PtpA of PtpA gene was successfully constructed, transfected into HEK293T and analyzed by SDS-PAGE. The specific molecular band was visible with relative molecular mass of 22 000.Western blot results showed that the expression product specifically bound to the primary antibody,demonstrating that the protein was a PtpA protein. In the double luciferase assay, the ratios of the relative fluorescence intensities of firefly luciferase and Renilla luciferase in the test group and the control group were significantly different from 2 to 24 h after transfection(P<0.05). The relative fluorescence values of firefly luciferase and Renilla luciferase in the control group were 2.93 times higher than those in the experimental group 2 h after transfection, indicating that PtpA protein had a significant inhibitory effect on the early activation of NF-κB signaling pathway. qPCR results showed that the expression levels of IL-6, GM-CSF,BIRC-2 and BIRC-3 in the control group were 3.93, 3.42, 2.17 and 2.30 times respectively of those in the test group 2 h after transfection(P<0.01), and were 4.26, 3.93, 2.36 and 2.50 times respectively of those in the test group 4 h after transfection(P<0.01). These results indicated that PtpA protein had a significant inhibitory effect on NF-κB signaling pathway-associated cytokines(IL-6, GM-CSF, BIRC-2 and BIRC-3) in the early stage of immunization.【Conclusion】qPCR results are consistent with the results of dual luciferase assay, indicating that the effect of M. bovis PtpA on NF-κB signaling pathway mainly occurs in the early stage. This study provides a theoretical basis for the follow-up study of effective tuberculosis prevention and control drugs.
【Objective】To investigate the effect of Mycobacterium bovis PtpA protein on NF-κB signaling pathway which is related to immune response, and reveal the role of PtpA protein in the body's response to immunity.【Method】The PtpA gene eukaryotic expression vector FLAG-PtpA was transfected into HEK293T for SDS-PAGE analysis and Western blot detection. After activation of NF-κB signaling pathway, the effects of PtpA protein on NF-κB signaling pathway were investigated by dual luciferase assay and qPCR.【Result】The eukaryotic expression vector FLAG-PtpA of PtpA gene was successfully constructed, transfected into HEK293T and analyzed by SDS-PAGE. The specific molecular band was visible with relative molecular mass of 22 000.Western blot results showed that the expression product specifically bound to the primary antibody,demonstrating that the protein was a PtpA protein. In the double luciferase assay, the ratios of the relative fluorescence intensities of firefly luciferase and Renilla luciferase in the test group and the control group were significantly different from 2 to 24 h after transfection(P<0.05). The relative fluorescence values of firefly luciferase and Renilla luciferase in the control group were 2.93 times higher than those in the experimental group 2 h after transfection, indicating that PtpA protein had a significant inhibitory effect on the early activation of NF-κB signaling pathway. qPCR results showed that the expression levels of IL-6, GM-CSF,BIRC-2 and BIRC-3 in the control group were 3.93, 3.42, 2.17 and 2.30 times respectively of those in the test group 2 h after transfection(P<0.01), and were 4.26, 3.93, 2.36 and 2.50 times respectively of those in the test group 4 h after transfection(P<0.01). These results indicated that PtpA protein had a significant inhibitory effect on NF-κB signaling pathway-associated cytokines(IL-6, GM-CSF, BIRC-2 and BIRC-3) in the early stage of immunization.【Conclusion】qPCR results are consistent with the results of dual luciferase assay, indicating that the effect of M. bovis PtpA on NF-κB signaling pathway mainly occurs in the early stage. This study provides a theoretical basis for the follow-up study of effective tuberculosis prevention and control drugs.
引文
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[17]WONG D,BACH H,SUN J,et al.Mycobacterium tuberculosis protein tyrosine phosphatase(PtpA)excludes host vacuolar-H+-ATPase to inhibit phagosome acidification[J].P roc Natl Acad Sci USA,2011,108(48):19371-19376.
[18]WANG J,GE P,QIANG L,et al.The mycobacterial phosphatase PtpA regulates the expression of host genes and promotes cell proliferation[J].Nat Commun,2017,8(1):244.
[19]孟露萍,史梦婷,包海洋,等.结核分枝杆菌Rv2626c蛋白对RAW264.7细胞凋亡的影响[J].中国畜牧兽医,2016,43(4):892-898.
[20]VOLKMAN H E,POZOS T C,ZHENG J,et al.Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium[J].Science,2010,327(5964):466-469.
[2]DELAHAY R J,DE LEEUW A N,BARLOW A M,et al.The status of Mycobacterium bovisinfection in UKwild mammals:A review[J].Vet J,2002,164(2):90-105.
[3]MACIEL A,LOIKO M R,BUENO T S,et al.Tuberculosis in Southern Brazilian wild boars(Sus scrofa):First epidemiological findings[J].Transbound Emerg Dis,2018,65(2):518-526.
[4]PHILLIPS C J C,FOSTER C R W,MORRIS P A,et al.The transmission of Mycobacterium bovis infection to cattle[J].Res Vet Sci,2003,74(2):1-15.
[5]PUCKEN V B,KNUBBEN-SCHWEIZER G,DOPFERD,et al.Evaluating diagnostic tests for bovine tuberculosis in the southern part of Germany:A latent class analysis[J].PLoS One,2017,12(6):e0179847.
[6]DE KANTOR I N and RITACCO V.An update on bovine tuberculosis programmes in Latin American and Caribbean countries[J].Vet Microbiol,2006,112(2/3/4):111-118.
[7]STONE M J,BROWN T J and DROBNIEWSKI F A.Human Mycobacterium bovis infections in London and Southeast England[J].J Clin Microbiol,2012,50(1):164-165.
[8]COWLEY S C,BABAKAIFF R and AV-GAY Y.Expression and localization of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA[J].Res Microbiol,2002,153(4):233-241.
[9]CHAO Y,XING Y,CHEN Y,et al.Structure and mechanism of the phosphotyrosyl phosphatase activator[J].Mol Cell,2006,23(4):535-546.
[10]WANG J,LI B X,GE P P,et al.Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system[J].Nat Immunol,2015,16(3):237-245.
[11]PFAFFI M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Res,2001,29(9):e45.
[12]TONKS N K.Protein tyrosine phosphatases:From genes,to function,to disease[J].Nat Rev Mol Cell Biol,2006,11(7):833-846.
[13]FU Y and GALAN J E.The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton[J].Mol Microbiol,1998,27(2):359-368.
[14]GOEBEL-GOODY S M,WILSON-WALLIS E D,ROY-STON S,et al.Genetic manipulation of STEP reverses behavioral abnormalities in a fragile X syndrome mouse model[J].Genes Brain Behav,2012,11(5):586-600.
[15]SHI L,POTTS M and KENNELLY P J.The serine,threonine,and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms:A family portrait[J].FEMS Microbiol Rev,1998,22(4):229-253.
[16]YONGFANG Z,KURUP P,JIAN X,et al.Genetic reduction of striatal-enriched tyrosine phosphatase(STEP)reverses cognitive and cellular deficits in an Alzheimer's disease mouse model[J].Proc Natl Acad Sci USA,2010,107(44):19014-19019.
[17]WONG D,BACH H,SUN J,et al.Mycobacterium tuberculosis protein tyrosine phosphatase(PtpA)excludes host vacuolar-H+-ATPase to inhibit phagosome acidification[J].P roc Natl Acad Sci USA,2011,108(48):19371-19376.
[18]WANG J,GE P,QIANG L,et al.The mycobacterial phosphatase PtpA regulates the expression of host genes and promotes cell proliferation[J].Nat Commun,2017,8(1):244.
[19]孟露萍,史梦婷,包海洋,等.结核分枝杆菌Rv2626c蛋白对RAW264.7细胞凋亡的影响[J].中国畜牧兽医,2016,43(4):892-898.
[20]VOLKMAN H E,POZOS T C,ZHENG J,et al.Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium[J].Science,2010,327(5964):466-469.