CAJ | 학술논문

目的：为检测环境中可能存在的致癌物,建立生长阻滞和 DNA 损伤诱生蛋白45(GADD45α)双荧光素报告基因系统,构建新型人 GADD45α荧光素酶报告基因质粒。方法在 GADD45α基因启动子序列2.2 kb 及其后基因组 DNA 序列2.6 kb 范围内,设计3对引物进行搭桥 PCR,获得全长为4924 bp 的目的序列,插入 pGL4.10[luc2]质粒相应位点内。质粒构建成功后测定全长并进行酶切鉴定。结果正确扩增了全长为4.9 kb 的人 GADD45α基因片段,成功构建了人 GADD45α报告基因质粒(pGD2.2K-luc2)。结论在质粒设计中加入了存在 p53非依赖性 BRCA1结合位点的 GADD45α基因第1个内含子,构建成功的 pGD2.2K-luc2,为转染人源性细胞建立 GADD45α双荧光素报告基因系统打下了基础。
목적：위검측배경중가능존재적치암물,건립생장조체화 DNA 손상유생단백45(GADD45α)쌍형광소보고기인계통,구건신형인 GADD45α형광소매보고기인질립。방법재 GADD45α기인계동자서렬2.2 kb 급기후기인조 DNA 서렬2.6 kb 범위내,설계3대인물진행탑교 PCR,획득전장위4924 bp 적목적서렬,삽입 pGL4.10[luc2]질립상응위점내。질립구건성공후측정전장병진행매절감정。결과정학확증료전장위4.9 kb 적인 GADD45α기인편단,성공구건료인 GADD45α보고기인질립(pGD2.2K-luc2)。결론재질립설계중가입료존재 p53비의뢰성 BRCA1결합위점적 GADD45α기인제1개내함자,구건성공적 pGD2.2K-luc2,위전염인원성세포건립 GADD45α쌍형광소보고기인계통타하료기출。
Objective To establish a genic system for GADD45α dual luciferase reporter (growth arrest DNA damage45α) and construct a new human GADD45α-luciferase reporter genes plasmid in order to detect the possible car-cinogens present in the environment. Methods Bridge polymerase chain reaction (PCR) was designed with three pairs of primers at the range from human GADD45α gene subsequence covering 2. 2kb to the following 2. 6kb genomic DNA se-quences. The full length of 4924 bp sequence was inserted in the corresponding site of pGL4. 10 [luc2] plasmid. After establishing the plasmid construction successfully, full-length sequencing was detected, and endonuclease digestion was i-dentified. Results Full length 4. 9 kb of human GADD45α gene fragment was correctly amplified, and the GADD45α-luciferase reporter plasmid (pGD2. 2k-luc2) was successfully constructed. Conclusion The plasmid pGD2. 2k-luc2 is successfully established by using plasmid with addition of the first intron of the human GADD45α gene, where a p53-in-dependent BRCA1 binding site is occupied, and it builts the foundation for establishing the human Gadd45α dual lucifer-ase reporter gene system.