论文部分内容阅读
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。
In order to obtain the fusion protein of adenovirus receptor sCAR and PPAb, the PPAb gene was cloned into the constructed prokaryotic expression vector pQE30-sCAR. The correct recombinant plasmid was identified by PCR and sequencing pQE30-sCAR-PPAb, obtained after transformation E. coli M15 engineered bacteria. After induced by IPTG, the strain highly expressed the fusion protein sCAR-PPAb with histidine tag as inclusion body. The inclusion bodies were denatured by urea, refolded with PBS and purified by Ni ion affinity chromatography to obtain the target protein. SDS-PAGE and Western blotting analysis showed that there was an obvious specific protein band around 42 kD. At the same time, the cell experiments showed that the fusion protein sCAR-PPAb can enhance the infection efficiency of Kasumi-1 by Ad-EGFP.