重组法国梧桐花粉过敏原2(rPla a2)对诱导型一氧化氮合酶(NOS2)基因启动子活性及基因表达的影响
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摘要
目的:构建人诱导型一氧化氮合酶(NOS2)基因启动子区,对NOS2启动子序列进行鉴定并分析其活性;探索重组法国梧桐花粉过敏原2(r Pla a2)对人NOS2基因启动子活性及基因表达的影响。方法:利用PCR扩增、限制性内切酶双酶切、DNA连接酶连接、大肠杆菌转化等方法将NOS2基因启动子区克隆至pGL3-Basic载体上,将所构建重组报告质粒转染至人胚肾(HEK293T)细胞中,利用双荧光素酶活性分析检测该重组报告质粒启动子活性及检测rPla a2对人NOS2基因启动子活性的影响;用实时荧光定量PCR法检测rPla a2对人NOS2基因mRNA水平的影响。结果:成功构建含有NOS2启动子的重组报告质粒,与对照(p GL3-Basic)组相比,含NOS2启动子区的重组质粒转染组荧光素酶活性显著增高;与不加刺激(NOS2)组相比,加入rPla a2刺激后含NOS2启动子区的重组报告质粒转染组荧光素酶活性显著增高,加入rPla a2刺激后NOS2 mRNA相对表达水平显著增高。结论:rPla a2可增强人NOS2基因启动子活性,并增加其基因表达。
Objective: To clone the promoter and identify the promoter activity in human inducible nitric oxide synthase( NOS2) gene and to identify the effect of recombinant platanus pollen allergen 2( rPla a2) on the promoter activity of human NOS2 gene in HEK293 T cells,and transcription and expression of human NOS2 gene in Beas-2 B cells. Methods: PCR,restricted enzyme,DNA ligase and E. coli were used to clone the promoter region of NOS2 gene into pGL3-Basic to construct a new luciferase reporter plasmid. Dual-luciferase reporter assay was used to identify the promoter activity and to measure the influence of r Pla a2 on NOS2 promoter activity. Quantitative Real-time PCR analysis was used to detect the influence of r Pla a2 on NOS2 expression in mRNA level. Results: The reporter plasmid that contained NOS2 promoter had been constructed successfully and been identified with significant luciferase activity compared with p GL3-Basic. NOS2 luciferase activity was significantly promoted by rPla a2 compared with blank control group( NOS2 without r Pla a2 stimulating group). rPla a2 induced mRNA expression of NOS2 compared with blank control group( NOS2 without r Pla a2 stimulating group). Conclusion: The expression of human NOS2 could be up-regulated by rPla a2 both in mRNA level and promoter level.
Objective: To clone the promoter and identify the promoter activity in human inducible nitric oxide synthase( NOS2) gene and to identify the effect of recombinant platanus pollen allergen 2( rPla a2) on the promoter activity of human NOS2 gene in HEK293 T cells,and transcription and expression of human NOS2 gene in Beas-2 B cells. Methods: PCR,restricted enzyme,DNA ligase and E. coli were used to clone the promoter region of NOS2 gene into pGL3-Basic to construct a new luciferase reporter plasmid. Dual-luciferase reporter assay was used to identify the promoter activity and to measure the influence of r Pla a2 on NOS2 promoter activity. Quantitative Real-time PCR analysis was used to detect the influence of r Pla a2 on NOS2 expression in mRNA level. Results: The reporter plasmid that contained NOS2 promoter had been constructed successfully and been identified with significant luciferase activity compared with p GL3-Basic. NOS2 luciferase activity was significantly promoted by rPla a2 compared with blank control group( NOS2 without r Pla a2 stimulating group). rPla a2 induced mRNA expression of NOS2 compared with blank control group( NOS2 without r Pla a2 stimulating group). Conclusion: The expression of human NOS2 could be up-regulated by rPla a2 both in mRNA level and promoter level.
引文
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[24]张广洋,郭耀杰,孙妍妍,等.GATA3-IL-13通路基因多态性和IL-13水平与哮喘病的相关性研究[J].中国免疫学杂志,2017,33(12):1849-1853.Zhang GY,Huo YJ,Sun YY,et al.GATA-IL-13 pathway gene polymorphisms are associated with serum IL-13 and asthma[J].Chin J Immunol,2017,33(12):1849-1853.
[25]Fernandez-gonzalez D,Gonzalez-parrado Z,Vega-maray AM,et al.Platanus pollen allergen,Pla a 1:quantification in the atmosphere and influence on a sensitizing population[J].Clin Exp,2010,40(11):1701-1708.
[26]Fernandez-gonzalez M,Guedes A,Abreu I,et al.Pla a_1 aeroallergen immunodetection related to the airborne Platanus pollen content[J].Sci Total Envir,2013,(463-464):855-860.
[2]Hu D.Role of Anti-inflammatory cytokines IL-35 and IL-37 in asthma[J].Inflammation,2017,40(2):697-707.
[3]Doreswamy V,Peden DB.Modulation of asthma by endotoxin[J].Clin Exp Allergy,2011,41(1):9-19.
[4]Natarajan S,Kim J,Bouchard J,et al.Pulmonary endotoxin tolerance protects against cockroach allergen-induced asthma-like inflammation in a mouse model[J].Int Archives Allergy Immunol,2012,158(2):120-130.
[5]Finkelman FD,Hogan SP,Hershey GK,et al.Importance of cytokines in murine allergic airway disease and human asthma[J].JImmunol,2010,184(4):1663-1674.
[6]Asam C,Hofer H,Wolf M,et al.Tree pollen allergens-an update from a molecular perspective[J].Allergy,2015,70(10):1201-1211.
[7]Ibarrola I,Arilla MC,Martinez A,et al.Identification of a polygalacturonase as a major allergen(Pla a 2)from Platanus acerifolia pollen[J].J Allergy Clin Immunol,2004,113(6):1185-1191.
[8]Rojo J,Rapp A,Lara B,et al.Characterisation of the airborne pollen spectrum in Guadalajara(central Spain)and estimation of the potential allergy risk[J].Environmental Monitoring Assessment,2016,188(3):130.
[9]Chibana K,Trudeau JB,Mustovich AT,et al.IL-13 induced increases in nitrite levels are primarily driven by increases in inducible nitric oxide synthase as compared with effects on arginases in human primary bronchial epithelial cells[J].Clin Exp Allergy,2008,38(6):936-946.
[10]Lane C,Knight D,Burgess S,et al.Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath[J].Thorax,2004,59(9):757-760.
[11]Roos AB,Mori M,Gronneberg R,et al.Elevated exhaled nitric oxide in allergen-provoked asthma is associated with airway epithelial i NOS[J].PLo S One,2014,9(2):e90018.
[12]Sato S,Wang X,Saito J,et al.Exhaled nitric oxide and inducible nitric oxide synthase gene polymorphism in Japanese asthmatics[J].Allergol Int,2016,65(3):300-305.
[13]Bastain TM,Islam T,Berhane KT,et al.Exhaled nitric oxide,susceptibility and new-onset asthma in the Children's Health Study[J].Eur Respir J,2011,37(3):523-531.
[14]Olin AC,Rosengren A,Thelle DS,et al.Increased fraction of exhaled nitric oxide predicts new-onset wheeze in a general population[J].Am J Respir Crit Care Med,2010,181(4):324-327.
[15]Malinovschi A,Fonseca JA,Jacinto T,et al.Exhaled nitric oxide levels and blood eosinophil counts independently associate with wheeze and asthma events in National Health and Nutrition Exa mination Survey subjects[J].J Allergy Clin Immunol,2013,132(4):821-827.
[16]Yamamoto M,Tochino Y,Chibana K,et al.Nitric oxide and related enzymes in asthma:relation to severity,enzyme function and inflammation[J].Clin Exp Allergy,2012,42(5):760-768.
[17]Cowan DC,Taylor DR,Peterson LE,et al.Biomarker-based asthma phenotypes of corticosteroid response[J].J Allergy Clin Immunol,2015,135(4):877-883.
[18]Larsson-callerfelt AK,Weitoft M,Nihlberg K,et al.i NOS affects matrix production in distal lung fibroblasts from patients with mild asthma[J].Pulm Pharmacol Ther,2015,34:64-71.
[19]Forstermann U,Sessa WC.Nitric oxide synthases:regulation and function[J].Eur Heart J,2012,33(7):829-837.
[20]El-gayar S,Thuring-nahler H,Pfeilschifter J,et al.Translational control of inducible nitric oxide synthase by IL-13 and arginine availability in inflammatory macrophages[J].J Immunol,2003,171(9):4561-4568.
[21]Choi WS,Seo YB,Shin PG,et al.Veratric acid inhibits i NOS expression through the regulation of PI3K activation and histone acetylation in LPS-stimulated RAW264.7 cells[J].Int J Mol Med,2015,35(1):202-210.
[22]Caramori G,Casolari P,Adcock I.Role of transcription factors in the pathogenesis of asthma and COPD[J].Cell Commun Adhesion,2013,20(1-2):21-40.
[23]幸鹏,刘志刚,喻海琼,等.表观遗传机制和哮喘的发生[J].中国免疫学杂志,2016,75(5):757-759,763.Xing P,Liu ZG,Yu HQ,et al.Epigenetic mechanism and asthma[J].Chin J Immunol,2016,75(5):757-759,763.
[24]张广洋,郭耀杰,孙妍妍,等.GATA3-IL-13通路基因多态性和IL-13水平与哮喘病的相关性研究[J].中国免疫学杂志,2017,33(12):1849-1853.Zhang GY,Huo YJ,Sun YY,et al.GATA-IL-13 pathway gene polymorphisms are associated with serum IL-13 and asthma[J].Chin J Immunol,2017,33(12):1849-1853.
[25]Fernandez-gonzalez D,Gonzalez-parrado Z,Vega-maray AM,et al.Platanus pollen allergen,Pla a 1:quantification in the atmosphere and influence on a sensitizing population[J].Clin Exp,2010,40(11):1701-1708.
[26]Fernandez-gonzalez M,Guedes A,Abreu I,et al.Pla a_1 aeroallergen immunodetection related to the airborne Platanus pollen content[J].Sci Total Envir,2013,(463-464):855-860.