SARS冠状病毒S受体结合区蛋白片段在杆状病毒载体中的表达及其抗原性研究

Expression of the Receptor-Binding Region of SARS-CoV Spike Protein in Baculovirus Vector and Identifying its Antigenicity

目的:利用Bac-to-Bac1杆状病毒系统,在sf9昆虫细胞中表达严重急性呼吸综合征(SARS)冠状病毒(SARS-CoV)的S受体结合区蛋白片段,并对其免疫原性进行研究。方法:将S蛋白的受体结合区基因片段定向克隆至转座载体pFast-Bac1,转化大肠杆菌DH10Bac感受态细胞,用抗生素平板筛选重组杆粒。脂质体介导重组杆粒转染sf9昆虫细胞,待细胞形态明显改变后收获细胞和培养上清液。利用SARS病人恢复期抗血清做ELISA和Western印迹,分析重组蛋白的抗原性。结果:ELISA和Western印迹表明,在sf9昆虫细胞中表达的SARS-CoVS受体结合区重组蛋白可与SARS病人恢复期抗血清发生特异反应。结论:获得了在昆虫细胞内表达的SARS-CoVS受体结合区重组蛋白,并证明该蛋白有可能用于SARS感染的抗体检测,为SARS-CoV免疫机制及其疫苗的进一步研究奠定了基础。

Objective： To express the receptor-binding region of SARS-CoV spike protein in sf9 insect cells by using Bac-to-Bacl vector and indentify its antigenicity. Methods： Recombinant plasmid pFastBacl/S was constructed by insert- ing the gene of receptor-binding region of SARS-CoV S protein into transposing vector pFastBacl, and pFastBacl/S plas- mid was further transformed into E.coli DH10Bac competent cells. The recombinant bacmid was screened by LB agar plate containing 3 kind of antibiotics. Fresh sf9 insect cells were transfected with the recombinant bacmid via lipofectin. Cells and cell supernatants were harvested separately when an obvious pathological changes could be observed. The recombinant protein was identified with SARS convalescent sera by Western blot and ELISA. Results： ELISA and Western blot analysis showed that the recombinant protein was able to react with all sera of SARS patients, which revealed antigen specificity of the recombinant protein. Conclusions： The recombinant protein can be expressed in sf9 insect cells successfully, and is possible for clinical application in antibody detection of SARS infection. It may provide a good strategy for not only further research of immune response to SARS-CoV but also further development of SARS vaccine.