含B7-H1启动子的荧光素酶报告基因载体的构建及-第三军医大学学报.DOC

B7-H1启动子荧光素酶报告基因载体的构建及活性检测 吴胜昔1，2，陈永文2，朱广倍1，费蕾2，吴玉章2* （1、400054 重庆 巴南，重庆理工大学药学与生物工程学院；2、400017 重庆 沙坪坝，第三军医大学基础部免疫学教研室） [摘要] 目的 PCR扩增B7-H1启动子基因，构建含B7-H1启动子的荧光素酶报告基因载体，转染肝癌HepG2细胞进行活性检测。方法 PCR扩增B7-H1启动子上游区域基因片段，并插入到含有荧光素酶报告基因的载体pGL3-Basic中构建重组子，经双酶切和测序鉴定后，将重组子转染HepG2细胞，采用双荧光素酶报告基因检测系统检测IFN-γ作用前后荧光素酶表达活性的变化。结果 通过酶切鉴定和基因测序，成功地构建了6种不同长度的pGL3-B7-H1-Luc荧光素酶表达质粒，分别命名为p88，p175, p203, p398, p723, p960。瞬时转染HepG2后经报告基因检测，p175, p203, p398, p723, p960所含五段启动子均具有转录活性，IFN-γ作用后启动子活性明显增强，而p88转染组IFN-γ作用后，荧光素酶活性无显著变化。结论 成功构建了B7-H1启动子的荧光素酶报告基因载体，为进一步探讨B7-H1启动子活性及其分子机制的研究奠定了基础。 [关键词] B7-H1；启动子；双荧光素酶报告基因检测；活性测定 Construction of luciferase reporter gene vectors containing B7-H1 promoter region and promoter activity assay WU Sheng-xi1，2，CHEN Yong-wen2，ZHU Guang-bei1，FEI Lei2, WU Yu-zhang2 School of Pharmacy and Bioengineering, Chongqing University of Technology，Chongqing 400050，China； 2. Department of immunology, Third Military Medical University , Chongqing 400017, China ) [Abstract] Objective B7-H1 gene promoters were amplified by PCR，to construct luciferase reporter gene vectors containing B7-H1 promoter region，and detect promoter activity in HepG2 cells. Methods Firstly several fragments of the B7-H1 gene 5’-UTR promoters were amplified by PCR and cloned into pGL3 basic vector. After the identification by digestion and sequencing on the recombinant basic vector, they were named p88，p175, p203, p398, p723, and p960 respectively. Then one of the human HCC cell lines, HepG2 cells, was transfected with these constructed plasmids. The expression of luciferase before and after the addition of IFN-γ was detected by Dual-Luciferase(LUC) Reporter Assay System kit. Results Five luciferase reporter gene vectors containing B7-H1 promoter region were constructed successfully identified by restriction endonuclease analysis and sequences analysis. The recombinant Constructs were transiently transfected into HepG2 cells. After 48h of transfection, the results of Dual-luciferase reporter gene assay showed