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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∶10 880,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。
OBJECTIVE: To prepare recombinant spores expressing SARS-CoV S protein receptor binding domain (RBD) using the Bacillus subtilis presentation technique. Methods: The B. subtilis CotB gene was constructed into the genome-integrated plasmid pDG1664. The RBD gene was ligated to the downstream of CotB gene to construct the recombinant plasmid pDG1664-CotB-RBD. The recombinant plasmid pDG1664-CotB-RBD was integrated into PY-79 Bacillus subtilis genome by homologous recombination. Recombinant bacteria were screened by erythromycin resistance and identified by PCR and DNA sequencing. Western blotting was used to identify the expression of RBD protein on the spores of the recombinant bacteria. Mice were immunized orally with recombinant spores expressing RBD and analyzed by ELISA and flow cytometry The immunogenicity of recombinant spores was tested. Results: The recombinant strain RS1931 with the RBD antigen gene was constructed in the Bacillus subtilis genome. The recombinant spore was expressed in the CotB-RBD fusion protein with a relative molecular mass of about 62 × 103. The RBD antigen-specific IgG antibody in the recombinant spleen-immunized mice Degrees up to 1:10 880 two weeks after the last immunization, and the proportion of IFN-γ + CD4 +, IL-4 + CD4 + and IFN-γ + CD8 + T cells in splenocytes of mice after 18 weeks of recombinant spleen priming was up-regulated, Oral immunization of spores produces good humoral and cellular immune responses. CONCLUSION: A recombinant Bacillus subtilis sporulation method was established based on the SARS coronavirus S protein RBD, and the recombinant RBD protein was successfully expressed on the surface of Bacillus subtilis spores. The obtained recombinant spores have good immunogenicity for the development of spore presentation SARS vaccine laid the foundation.