乏氧／辐射双敏感启动子介导分泌型人TRAIL基因载体的构建及表达  

Construction and expression of secreting type human TRAIL gene vector mediated by hypoxia/radiation double sensitive promoter

作　　者：杨艳明[1] 李艳博[2] 贾晓晶[1] 曲雅勤[1] 

机构地区：[1]吉林大学第二医院放疗科,吉林长春130041 [2]吉林大学公共卫生学院卫生部放射生物学重点实验室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2009年第3期431-436,共6页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅白求恩基金资助课题(200705221)

摘　　要：目的:构建乏氧(HRE)/辐射(Egr1)双敏感启动子介导的分泌型人TRAIL(shTRAIL)基因表达载体pcDNA3.1-HRE-Egr1-shTRAIL,观察乏氧和辐射对其表达的影响。方法:利用化学合成HRE上、下链,PCR扩增得到双链HRE;pMD19T-Egr1经SacⅠ和HindⅢ双酶切得到Egr1;pshuttle-shTRAIL经KpnⅠ和BamHⅠ双酶切得到shTRAIL;利用基因重组技术构建含有HRE/Egr1双敏感启动子介导shTRAIL的表达载体pcDNA3.1-HRE-Egr1-shTRAIL,经酶切、PCR和测序鉴定正确。肺腺癌A549细胞分为正常组、乏氧组(1%)、照射组(6 Gy)和乏氧加照射组;表达载体转染肺腺癌A549细胞,RT-PCR及ELISA法检测shTRAIL的表达。结果:BamHⅠ和SmaⅠ酶切,所得片段大小分别为1 284和4 998 bp、2 292和3 990 bp;以Egr1和shTRAIL引物PCR扩增该载体,得到469和820 bp产物;将pcDNA3.1-HRE-Egr1-shTRAIL进行测序,所得结果与设计一致,说明构建正确。转染肺腺癌A549细胞后,乏氧组、辐射组及乏氧加照射组shTRAIL mRNA和蛋白表达增加,与对照组比较差异有显著性(P<0.05),且乏氧加照射组表达更明显。结论:成功地构建了乏氧/辐射双敏感启动子介导分泌型人TRAIL基因表达的载体pcDNA3.1-HRE-Egr1-shTRAIL;乏氧及照射能够诱导TRAIL表达增加,且二者具有协同作用。Objective To construct secreting type human TRAIL （shTRAIL） gene vector pcDNA3.1-HRE/Egr1- shTRAIL mediated by hypoxia/radiation double sensitive promoter, and observe the effect of hypoxia and radiation on shTRAIL. Methods HRE upper and lower strands were gotten by chemical synthesis, double strands HRE was gotten by PCR; pMD19T-Egrl was digested by Sac I and Hind Ⅲ, then Egrl was obtained, pshuttle-shTRAIL was digested by Kpn I and BarnH I, then shTRAIL was obtained; HRE/Egrl double sensitive promoter mediated shTRAIL expression vector pcDNA3.1-HRE/Egr1-shTRAIL was constructed by gene recombination technique, it was identified correctlly by enzyme digestion, PCR and sequencing. A549 cells were divided into normal, hypoxia （0.1%）, irradiation （6 Gy） and hypoxia ＋ irradiation groups. Results After enzyme digestion by BamH I and Sma I , the fragments which lengthes were 1284 bp and 4 998 bp, 2 292 bp and 3 990 bp were obtained; the vector was amplified by PCR with Egrl and shTRAIL primer, the products which lengthes were 469 bp and 820 bp were obtained; pcDNA3.1-HRE/Egr1-shTRAIL was sequenced, the result was same to designed, this demonstrated that the construction was right. The vectors were transfected into A549 cells of adenocarcinoma of lung, the expression levels of shTRAIL mRNA and protein were increased after treated with hypoxia and radiation, it had statistically significant differences compared with normal group （P〈0.05）, and when they were combinated, the effect was more obvious. Conclusion Secreting type human TRAIL gene vector pcDNA3. 1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter is constructed successfully, and hypoxia and radiation could increase the expression of TRAIL, and they have synergetic effect.

关 键 词：乏氧/辐射 肿瘤坏死因子相关凋亡诱导配体 基因重组 

分 类 号：Q78[生物学—分子生物学] Q691