小鼠穿孔素在杆状病毒表达系统中的克隆与表达

Clone and Expression of Rat Perforin in Baculovirus System

穿孔素是机体发挥细胞毒作用的重要效应物质 ,在抗肿瘤、抗病毒及寄生虫治疗中有着重要的意义 ,但它在动物体内的含量极少 ,不便于进一步研究及新药开发 ,用基因工程方法来表达生产穿孔素是将小鼠的穿孔素基因在杆状病毒表达系统中进行克隆与表达。表达产物经SDS PAGE检测、薄层扫描确定表达量为细胞总蛋白的 2 5 % ,表达量在第 4d达到最高 ,其发挥溶红细胞活性的最佳Ca2 + 浓度为 2mmol/L。

It is known that perforin plays an important role in the treatment of cancer,virus and parasite. But,the extensive biochemical and structural/functional analysis and studies of perforin were restricted because this protein is produced by killer lymphocytes in only modest amounts. In this paper,rat perforin was expressed in insect cells using Baculovirus expression system. The activity of recombinant perforin was measured by lysis of erythrocytes using a hemolysis assay. A time course analysis revealed that the expression of perforin reached maximum at around 72～96 h. The amount of expression product comes to be approximately 25% of the total lysates through SDS PAGE. The interaction of Ca 2+ on perforin was also studied and the optimal concentration was 2 mmol/L.