LC-1ScFv偶联GFP基因的构建及表达

Construction and expression of fusion gene of LC-1 ScFv and GFP

从具有高效蛋白表达及活力的抗肺癌杂交瘤单克隆细胞株(LC-1)抽提总RNA,反转录成cDNA.根据肿瘤单链抗体(ScFv)基因设计引物,用多聚酶链式反应(PCR)克隆出抗体的重链和轻链基因,并通过重叠延伸PCR法将重链基因与修饰后的轻链基因连接为单链抗体基因.改造后的ScFv与绿色荧光蛋白(GFP)基因连接,构建表达质粒ProGFP-ScFv,随后转化大肠杆菌JM109,用诱导剂IPTG进行诱导表达.表达产物采用Ni-NTA树脂进行纯化,进行SDS-PAGE电泳分析其纯度.酶联免疫检测(ELISA)表明该蛋白已具有抗体活性.395nm激光激发显示绿色荧光,表明目的基因在原核生物中存在表达.

Total RNA of anti-lung cancer hybridoma cell line (LC-1), which has high secretory volume and activity, was isolated and transferred into cDNA. According to the gene of single chain fibroma virus (ScFv), the variable regions of heavy chain (VH) and light chain (VL) were cloned through polymerase chain reaction (PCR), respectively. ScFv gene was formed by the ligation of VH and modified VL using strain-overlapped elongating PCR, and fused with GFP gene. After inserting the fusion gene into pProEx HTb, the expression plasmid ProGFP-ScFv was constructed. The fusion protein was expressed in E. coli cell line JM109 with an induction of IPTG; and after the purification using Ni-NTA resin, it was analyzed by SDS-PAGE and ELISA. The host cells fluorescing bright green at 395 nm wavelength indicated that the expected fusion gene was expressed in the prokaryotic expression system.