Basic Research for Development of Expression Cloning Vectors Using Insect Cell Culture Systems

为了建立利用培养的昆虫细胞建立真核细胞基因表达克隆载体系统所需的基础技术，本研究构建了杆状病毒载体系统中辅助病毒控制下外源基因的表达克隆载体和果蝇S2细胞系统中共表达扑霉素抗性(PAC)基因的表达克隆载体，并获得了以下结果。在杆状病毒载体系统中，构建了含多角体蛋白启动子的表达克隆载体pBMIHpa(约5.8kbp)，插入外源基因后，与杆状病毒DNA共转染家蚕细胞(BmN4)，虽然共转细胞中检测到外源基因的表达，但由于病毒增殖导致细胞裂解，无法克隆细胞。针对S2细胞系统，将绿色荧光蛋白基因插入到表达克隆载体Vect…中More或pDhsp(pac^+)共表达pac基因作为选择标记或另一种缺失pac基因的质粒载体pDhsp(pac-)。PDhspGFP(pac^+)与pDhspGFP(pac^-)和pDhsp(pac^+)共转染获得的S2细胞的GFP荧光强度明显高于pDhspGFP(pac^+)转化的S2细胞，说明利用共表达载体可以有效地提高检测目的基因表达的灵敏度。将LacZ基因表达载体pDhspLacZ(pac^-)和pDhsp(pac^+)以1：1或1：10的比例共转染入S2细胞中，可检测到β-半乳糖苷酶的活性，但以1：100的比例不存在，提示共转染S2细胞的克隆数应在10~100个克隆之间。较少

In order to establish basic technologies required for development of eukatyotic gene expression cloning vector systems using cultured insect cells, I have constructed an expression cloning plasmid vector which expressed foreign genes under control of the helper virus for the baculovirus vector system and another expression cloning plasmid vector which coexpresses puromycin resistant (pac) gene for the Drosophila S2 cell system, and obtained following results.1. For the baculovirus vector system, the expression cloning plasmid vector pBMIHpa (ca. 5.8 kbp) containing the polyhedrin promoter was constructed and, after inserting foreign genes, cotransfected to the silkworm cells (BmN4) with the baculovirus DNA.Although the foreign gene expression was detected in the cotransfected cells, it was impossible to clone the cells because of the cell lysis by virus multiplication.2. For the S2 cell system, the green fluorescent protein (GFP) gene was inserted to the expression cloning plasmid vect … More or pDhsp(pac^+) coexpressing the pac gene as a selectable marker or another plasmid vector pDhsp(pac-) lacking the pac gene. The GFP fluorescence intensity in the transformed S2 cells, which were obtained by transfection with pDhspGFP(pac^+), was remarkably higher than that in another transformed S2 cells, which were obtained by cotransfection with pDhspGFP(pac^-) and pDhsp(pac^+), demonstrating that the use of coexpression plasmid vector can effectively enhance the sensitivity in the detection of the target gene expression. Detectable levels of beta-galactosidase activity were observed in transformed S2 cells by cotransfection of the LacZ gene expressing plasmid pDhspLacZ(pac^-) and pDhsp(pac^+) at 1 : 1 or 1 : 10 ratio, but not at 1 : 100 ratio, suggesting that the limit number of clone for screening the cotransfected S2 cells should be between 10 to 100 clones.According to the results obtained in this research, I will further investigate the development of efficient screening methods of transformed insect cells. Less