一种展示SARS冠状病毒受体结合区的重组枯草杆菌芽孢的制备及免疫原性分析

目的：利用枯草杆菌芽孢呈递技术制备表达SARS冠状病毒S蛋白受体结合区（RBD）的重组芽孢。方法：将枯草杆菌 CotB 基因构建到基因组整合质粒pDG1664中，再将 RBD 基因连接到 CotB 基因的下游，构建成重组质粒pDG1664-CotB-RBD，通过同源重组整合到PY-79枯草杆菌基因组中；利用红霉素抗性筛选重组菌并进行PCR和DNA测序鉴定，Western印迹鉴定重组菌芽孢表面RBD蛋白的表达情况；用表达RBD的重组芽孢以口服方式免疫小鼠，通过ELISA和流式细胞术检测重组芽孢的免疫原性。结果：制备出枯草杆菌基因组整合了RBD抗原基因的重组菌株RS1931，形成的重组芽孢表达相对分子质量约62×103的CotB-RBD融合蛋白；重组芽孢免疫的小鼠血清RBD抗原特异性IgG抗体滴度在末次免疫后2周可达1∶10880，重组芽孢初免后18周的小鼠脾细胞中IFN-γ＋CD4^＋、IL-4＋CD4^＋和IFN-γ＋CD8^＋T细胞比例上调，表明重组芽孢经口服免疫产生良好的体液免疫和细胞免疫应答。结论：针对SARS冠状病毒S蛋白RBD建立了枯草杆菌芽孢呈递技术方法，制备出在枯草杆菌芽孢表面稳定表达外源RBD蛋白的重组株，获得的重组芽孢具有良好的免疫原性，为开发芽孢呈递型SARS疫苗奠定了基础。

Preparation and Immunogenicity Analysis of a Recombinant Bacillus subtilis Spore Displaying Receptor Binding Domain of SARS-CoV

Objective： To prepare recombinant spore displaying receptor binding domain（RBD） of SARS-CoV＇s S protein based on the Bacillus subtilis spore surface-displaying technique. Methods： The CotB gene from B.subtilis was inserted into pDG1664 integrational vector followed by ligation with RBD gene in the downstream to construct recombinant plasmid pDG1664-CotB-RBD, then it was integrated to genome of B.subtilis PY-79 by homologous recombination. The recombinant strain was screened by erythromycin resistance and further identified by PCR and DNA sequencing. The expression of RBD protein on spore coat of the recombinant strain was detected by Western blot. Mice were subsequently vaccinated orally with recombinant spore expressing RBD and the immunogenieity of recombinant spore was measured by ELISA and floweytometry. Results： The recombinant B.subtilis strain RS1931 with the genome integrated with RBD gene was prepared, and it can form spores expressing about 62 kD of CotB-RBD fusion protein. The RBD-specific sera IgG antibody titer from mice vaccinated with recombinant spores reached 1：10 880 two weeks post-last boost. The IFN-γ＋CD4^＋, IL-4＋CD4^＋and IFN-γ＋CD8^＋ T cells in splenocytes of mice vaccinated with recombinant spores upgraded, which indicated the good humoral and cellular immunities induced by oral vaccination with recombinant spores. Conclusion： The technology displaying RBD of SARS-CoV＇s S protein on the surface of B.subtilis spore was established and spores forming from the recombinant B.subtilis strain can stably express RBD and have good immunogenicity. This work has laid a foundation for development of novel SARS vaccines in B.subtilis spore-displaying system.