抗人脑胶质瘤重组免疫毒素SZ39(ScFv)-PE40的基因构建及表达

Construction and expression of a recombinant immunotoxin against human glioma, SZ39(ScFv)-PE40

目的 构建抗人脑胶质瘤重组免疫毒素SZ39(ScFv) PE4 0并在大肠杆菌中表达。方法 从质粒pVC85中克隆出PE4 0基因 ,与抗胶质瘤单链抗体SZ39 ScFv基因进行拼接 ,构建出重组免疫毒素SZ39(ScFv) PE4 0基因 ,并将其克隆于表达载体pET2 0b(+) ,在大肠杆菌Bl2 1(DE3)pLysS中以IPTG诱导表达。结果 SDS PAGE分析显示表达产物主要以包涵体的形式存在 ,分子量为 6 8kD左右 ,与SZ39(ScFv) PE4 0的分子量理论推算值相符 ;Westernblot显示在 6 8kD处出现特异性显色印迹 ;凝胶灰度扫描显示其表达量占菌体总蛋白的 2 0 %。结论 重组免疫毒素SZ39(ScFv) PE4 0可经原核表达载体 ,pET2 0b(+)在大肠杆菌BL2 1(DE3)pLysS中以包涵体形式得到较高效率的表达。

Objective To construct the gene of recombinant immunotoxin SZ39(ScFv) PE40 and express it in E.coli. Methods The cDNA encoding PE40 was amplified by PCR in which the plasmid pVC85 served as template, then the PE40 gene was attached to SZ39 ScFv gene to construct the SZ39(ScFv) PE40 gene, and then it was inserted into expression vector pET20b(+). The expression of SZ39(ScFv) PE40 was induced by IPTG in E.coli BL21(DE3)pLysS. Results SDS PAGE showed a novel protein band with an anti^cipated molecular mass of 68kD, which could also be determined by Western blot assay. Scanning of SDS PAGE gel showed that the expressed protein accounted for 20% of the total bacterial protein, it existed in the form of inclusion body. Conclusion The recombinant immunotoxin SZ39(ScFv) PE40 can be highly expressed in E.coli BL21(DE3)pLysS via expression vector pET20b(+) in the form of inclusion body.