Liu Shui-ping, Tan De-ming, Hou Zhou-hua
Institute of Infectious Disease, Xiangya Hospital, Department of Microbiology, Xiangya School of Medicine, Central South University, Changsha 410008, China.
Hunan Yi Ke Da Xue Xue Bao. 2003 Feb 28;28(1):20-2.
To construct an expression vector with a bicistron of green fluorescent protein (GFP) and HCV 5'NCR controlled luciferase gene and to explore its expression in hepatocytes.
With molecular cloning techniques, the DNA fragment of HCV 5'NCR and its controlled luciferase gene was obtained from plasmid pCMVNCRLuc and subcloned into pEGFP-C1, an expression plasmid of GFP. A recombinant plasmid pGFPNCRLuc with a bicistron of GFP and HCV 5'NCR controlled luciferase gene was constructed, and liposome-mediated transient expression was detected in hepatocyte QSG7701 cells.
The recombinant plasmid expressed both GFP and luciferase at a high level. The expression activity of GFP showed no significant difference compared to that of pEGFP-C1 (P > 0.05), nor did that of luciferase to pCMVNCRLuc (P > 0.05).
An expression vector with a bicistron of GFP and HCV 5'NCR controlled luciferase gene was successfully constructed. GFP and luciferase could express independently. This study can promote the establishment of an anti-HCV drug screen system.
构建一个带有绿色荧光蛋白（GFP）双顺反子和丙型肝炎病毒（HCV）5'非编码区（5'NCR）调控的荧光素酶基因的表达载体，并探讨其在肝细胞中的表达情况。
采用分子克隆技术，从质粒pCMVNCRLuc中获取HCV 5'NCR及其调控的荧光素酶基因的DNA片段，并亚克隆至GFP表达质粒pEGFP-C1中。构建了带有GFP和HCV 5'NCR调控的荧光素酶基因双顺反子的重组质粒pGFPNCRLuc，并在肝细胞QSG7701细胞中进行脂质体介导的瞬时表达检测。
重组质粒能高水平表达GFP和荧光素酶。GFP的表达活性与pEGFP-C1相比无显著差异（P>0.05），荧光素酶的表达活性与pCMVNCRLuc相比也无显著差异（P>0.05）。
成功构建了带有GFP和HCV 5'NCR调控的荧光素酶基因双顺反子的表达载体。GFP和荧光素酶可独立表达。本研究有助于抗HCV药物筛选系统的建立。
