重组双荧光素酶报告基因质粒psiCHECK-2-Intron构建转染及转染细胞萤火虫荧光素酶和海肾荧光素酶表达  

Construction of recombinant dual-luciferase reporter gene plasmid psiCHECK-2-Intron and expression of firefly luciferase and renilla luciferase in non-small-cell lung cancer cell line PC-9 after transfection

作　　者：廖亮 王琴容 杨国辉 LIAO Liang;WANG Qinrong;YANG Guohui(The Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;不详)

机构地区：[1]贵州医科大学附属医院,贵阳550004 [2]贵州省医学分子生物学重点实验室

出　　处：《山东医药》2020年第9期32-35,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81660474)。

摘　　要：目的构建重组双荧光素酶报告基因质粒psiCHECK-2-Intron,并观察其在非小细胞肺癌细胞株PC-9转染后对萤火虫荧光素酶、海肾荧光素酶表达。方法以双荧光素酶报告基因质粒psiCHECK-2中的嵌合体内含子(设计长度为133 bp)为模板,PCR扩增后插入到psiCHECK-2质粒中,即获得重组双荧光素酶报告基因质粒psi-CHECK-2-Intron。取psiCHECK-2-Intron质粒,转染至感受态大肠杆菌中,采用琼脂糖凝胶电泳实验观察目的基因片段长度,并对目的基因片段进行基因测序。将重组双荧光素酶报告基因质粒psiCHECK-2-Intron、双荧光素酶报告基因质粒psiCHECK-2分别转染至PC-9细胞中,记为转染组、对照组,采用qRT-PCR法检测两组细胞中萤火虫荧光素酶mRNA、海肾荧光素酶mRNA,采用双荧光素酶活性实验检测两组细胞中萤火虫荧光素酶活性、海肾荧光素酶活性。结果目的基因片段长度为133 bp,基因序列正确,无突变和缺失,表明重组双荧光素酶报告基因质粒psiCHECK-2-Intron构建成功。转染组细胞中萤火虫荧光素酶mRNA、海肾荧光素酶mRNA的相对表达量分别为2.213±0.038、2.004±0.025,对照组分别为1.004±0.012、1.003±0.010,两组相比,P均<0.05。转染组细胞中萤火虫荧光素酶活性、海肾荧光素酶活性分别为2.974±0.099、1.948±0.052,对照组分别为1.000±0.018、1.000±0.020,两组相比,P均<0.05。结论成功构建了重组双荧光素酶报告基因质粒psiCHECK-2-Intron;psiCHECK-2-Intron转染PC-9细胞后,细胞中萤火虫荧光素酶、海肾荧光素酶的表达均升高。Objective To construct the recombinant dual-luciferase reporter gene plasmid psiCHECK-2-Intron and to observe the expression of firefly luciferase and renilla luciferase after transfection in non-small-cell lung cancer cell line PC-9.Methods Firstly,using the psiCHECK-2 plasmid as a template,the Intron target fragment(designed length 133bp)was amplified by PCR with suitable primers,and then it was inserted into the multiple cloning site of psiCHECK-2 plasmid to construct the recombinant plasmid of psiCHECK-2-Intron dual-luciferase reporter gene.Secondly,the psiCHECK-2-Intron plasmid was transfected into E.coli and the length of the target gene fragment was observed by agarose gel electrophoresis.The transfected positive samples were identified by gene sequencing in order to determine the integrity of the inserted target gene.Thirdly,the recombinant dual-luciferase reporter gene plasmid psiCHECK-2-Intron and dual-luciferase reporter gene plasmid psiCHECK-2 were transfected into PC-9 cells,respectively,which were recorded as the transfection group and control group.The mRNAs of firefly luciferase and renilla luciferase were detected by qRT-PCR and the dual-luciferase reporter gene assay was used to detect the activity of both luciferases.Results The target gene fragment was 133 bp in length,and the gene sequence was correct without mutations and deletions,indicating that the recombinant dual-luciferase reporter gene plasmid psiCHECK-2-Intron was constructed successfully.The relative mRNA expression levels of firefly luciferase and renilla luciferase were 2.213±0.038 and 2.004±0.025,respectively,in the transfection group,versus 1.004±0.012 and 1.003±0.010,respectively,in the control group,with statistically significant difference(both P<0.05).The firefly luciferase and renilla luciferase activities were 2.974±0.099 and 1.948±0.052,respectively,in the transfection group,versus 1.000±0.018 and 1.000±0.020,respectively,in the control group(both P<0.05).Conclusion The recombinant psiCHECK-2-Intron plasmid is con

关 键 词：荧光素酶报告基因 双荧光素酶报告基因 重组双荧光素酶报告基因 嵌合体内含子 基因质粒 萤火虫荧光素酶 海肾荧光素酶 细胞实验 

分 类 号：R734.2[医药卫生—肿瘤]