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  

Recombinant platanus pollen allergen 2 ( rPla a 2 ) up-regulates human NOS2 gene promoter activity and production

作　　者：乔笑芹 王进雅[2] 路康[1] 曹倩[1] 周国平[1] QIAO Xiao-Qin;WANG Jin-Ya;LU Kang;CAO Qian;ZHOU Guo-Ping(Department of Pediatrics,the First AffiliatedHospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院儿科,南京210029 [2]南京医科大学第二附属医院儿科,南京210000

出　　处：《中国免疫学杂志》2018年第8期1143-1147,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金(81500013);南京市科技计划项目(201503003)资助

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.

关 键 词：诱导型一氧化氮合酶(NOS2) 重组法国梧桐花粉过敏原2(rPla a2) 启动子 哮喘 

分 类 号：R392.8[医药卫生—免疫学]