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通过RTPCR从SARS病毒BJ01株的RNA中扩增全长N基因,经凝胶回收后插入pBAD/TOPOThioFusion表达载体。然后在Top10中利用阿拉伯糖进行诱导表达,在优化的条件下,表达产物以可溶性为主,表达量约占细菌可溶性总蛋白的47%。表达产物采用ProBond蛋白纯化系统纯化后,目的蛋白约占67%。经免疫印迹法检测,表达产物与SARS病人恢复期血清和兔的抗血清均可进行特异反应。BJ01株核壳蛋白在大肠杆菌中的高效可溶性表达为后续的ELISA试剂盒的研制奠定了基础。
The full-length N gene was amplified from the RNA of the SARS virus BJ01 strain by RTPCR, recovered by gel and inserted into the pBAD / TOPOThioFusion expression vector. Then, arabinose was used to induce the expression in Top10. Under optimal conditions, the expression product was mainly soluble, accounting for about 47% of the total bacterial soluble protein. The expressed product was purified with ProBond protein purification system, the target protein accounted for about 67%. Western blot detection, the expression product and SARS patients convalescent serum and rabbit antiserum can be specifically reacted. The efficient and soluble expression of BJ01 nucleocapsid protein in Escherichia coli laid the foundation for the development of the subsequent ELISA kit.