sCAR-PPAb基因的原核表达载体构建及重组蛋白的表达纯化

Construction of Prokaryotic Expression Vector and Prokaryotic Expression,Purification of sCAR-PPAb

为获得腺病毒受体sCAR与掌叶半夏蛋白(PPA)的亚基B(PPAb)的融合蛋白,将PPAb基因克隆入已构建的原核表达载体pQE30-sCAR中,经PCR和测序鉴定正确的重组质粒pQE30-sCAR-PPAb,转化大肠杆菌M15后获得工程菌。该菌株经IPTG诱导后,高效表达出带有组氨酸标签以包涵体形式存在的融合蛋白sCAR-PPAb。包涵体经过尿素变性溶解、PBS稀释复性、Ni离子亲和层析柱纯化,获得目的蛋白。SDS-PAGE及Western blotting分析表明,在42 kD左右有一条明显的特异性蛋白条带。同时细胞实验结果表明融合蛋白sCAR-PPAb能提高Ad-EGFP对Kasumi-1的感染效率。

To obtain a fusion protein containing soluble Coxsackie-adenovirus receptor （sCAR） and the domain B of Pinellia pedatisecta agglutinin （PPAb）, the expression vector pQE30-sCAR-PPAb is con- structed by inserting domain B of PPA gene into pQE30-sCAR which has constructed and is identified by PCR and sequence analysis, Then an expression strain is selected after transformation of the recombined plasmid into E. coli M15, fusion protein with His-tag is efficiently expressed in the form of inclusion body after IPTG induction. The inclusion body is washed, dissolved in renatured by the gradual removal of urea via dialysis in solubilization buffer and purified by Ni^＋2 chelate affinity chromatography under renatured condition. SDS-PAGE analysis and Western blotting with His-probe（mouse monoclonal IgG） show that the fusion protein with a molecular weight of about 42 is purified. Ad-EGFP transduction of leukemia cell lines Kasumi-1 is increased by sCAR-PPAb fusion protein, indicating that the purified protein is confirmed to be of bioactivity,