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用PCR扩增SARS冠状病毒N蛋白全长cDNA,克隆到酵母表达载体pPIC3·5K,构建pPIC3·5K_SCoVN酵母表达质粒。表达质粒线性化后电转化到毕赤酵母GS115中,经G418_RDB,MM/MD平板与PCR扩增筛选获得His+Mut+重组菌株。比较研究了不同的培养基、溶解氧以及甲醇浓度对菌株生长与重组蛋白表达的影响。结果表明:FBS培养基最适宜重组菌的生长与表达,溶氧对菌体的生长与表达有显著的影响,甲醇诱导最佳终浓度为1%(V/V),SDS_PAGE分析重组蛋白的表达量,发现重组N蛋白表达量占细胞总蛋白的6%,每升培养基可以生产410mg重组N蛋白,生物量达45OD600。Westernblotting结果表明,重组N蛋白对鼠源单克隆抗体以及SARS病人恢复期血清具有较强的特异性反应。对摇瓶培养条件进行了发酵罐放大实验,结果生物量达到348OD600,表达量达到2·5g/L,分别为摇瓶表达的7·7倍和6·1倍,为SARS早期血清学诊断研究以及为N蛋白在病毒复制以及致病机理的研究奠定了一定的基础。
The full length cDNA of SARS coronavirus N protein was amplified by PCR and cloned into the yeast expression vector pPIC3.5K to construct the yeast expression plasmid pPIC3.5K_SCoVN. After linearization, the expression plasmid was transformed into Pichia pastoris GS115. His + Mut + recombinant strain was obtained by G418_RDB, MM / MD plates and PCR amplification. The effects of different media, dissolved oxygen and methanol concentration on the growth of the strain and the expression of recombinant protein were compared. The results showed that FBS medium was most suitable for the growth and expression of recombinant bacteria. Dissolved oxygen had a significant effect on the growth and expression of the bacterial cells. The optimum final concentration of methanol was 1% (V / V), and the expression of recombinant protein was analyzed by SDS-PAGE The results showed that the expression of recombinant N protein accounted for 6% of the total cellular protein, which could produce 410mg recombinant N protein per liter of culture medium with a biomass of 45OD600. Western blotting results showed that the recombinant N protein had a strong specific response to murine monoclonal antibody and convalescent serum of SARS patients. The culture conditions of the flask were magnified in a fermentor. The results showed that the biomass reached 348 OD600 with an expression level of 2.5 g / L, which was respectively 7.7 times and 6.1 times higher than those in the shake flask. It was an early serological diagnosis of SARS As well as for the N protein in the virus replication and pathogenesis of the study laid a solid foundation.