【摘要】 根据猪捷申病毒(PTV)Swine/CH/IMH/03株的基因组序列(GenBank登录号:DQ355222)设计了1对引物,采用RT-PCR方法扩增出了PTV的VP1基因片段,将其克隆到表达载体pET-32a(+)中,构建了重组质粒pET-VP1,经测序鉴定正确后,将其转化感受态细胞BL21(DE3)中,并进行IPTG诱导表达。结果表明,重组菌可表达分子质量为45 ku的融合蛋白,在1.0 mmol/L IPTG诱导6 h后,重组菌的表达效果最好,目的蛋白表达量占菌体总蛋白的63.4%,表达的蛋白以包涵体的形式存在于菌体中。表达产物经Ni柱纯化后,薄层扫描显示,目的蛋白的纯度达到了97%。Western-blot结果显示,纯化后的VP1蛋白能与PTV Swine/CH/IMH/03株阳性血清发生特异性的免疫印迹反应,表明,原核表达的VP1蛋白具有良好的反应原性。更多还原

【Abstract】 One pair of primers was designed according to the published sequences of VP1 gene of porcine teschovirus Swine/CH/IMH/03 strain.The VP1 gene was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-32a(+) to construct a recombinant pET-VP1.After sequencing,the recombinant was transformed into Escherichia coli BL21(DE3) competent cells.The transformed bacteria were induced by IPTG to produce a recombinant protein of 45ku of molecular weight.The result showed that 1.0mmol/L IPTG and 6h of induction time were the best conditions for VP1 protein production and the expressed protein accounted for 63.4% of total proteins.The SDS-PAGE analysis and thin-layer scanning of the purified protein showed that the purity of VP1 protein reached 97%.The purified protein could react with the positive serum with the antibody against strain Swine/CH/IMH/03 in a Western-blot test,indicating that the expressed protein possessed strong reactinogenicity.更多还原