CAJ | 학술논문

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。
목적：이용고초간균아포정체기술제비표체SARS관상병독S단백수체결합구（RBD）적중조아포。방법：장고초간균CotB기인구건도기인조정합질립pDG1664중，재장RBD기인련접도CotB기인적하유，구건성중조질립pDG1664-CotB-RBD，통과동원중조정합도PY-79고초간균기인조중；이용홍매소항성사선중조균병진행PCR화DNA측서감정，Western인적감정중조균아포표면RBD단백적표체정황；용표체RBD적중조아포이구복방식면역소서，통과ELISA화류식세포술검측중조아포적면역원성。결과：제비출고초간균기인조정합료RBD항원기인적중조균주RS1931，형성적중조아포표체상대분자질량약62×103적CotB-RBD융합단백；중조아포면역적소서혈청RBD항원특이성IgG항체적도재말차면역후2주가체1∶10880，중조아포초면후18주적소서비세포중IFN-γ+CD4+、IL-4+CD4+화IFN-γ+CD8+ T세포비례상조，표명중조아포경구복면역산생량호적체액면역화세포면역응답。결론：침대SARS관상병독S단백RBD건립료고초간균아포정체기술방법，제비출재고초간균아포표면은정표체외원RBD단백적중조주，획득적중조아포구유량호적면역원성，위개발아포정체형SARS역묘전정료기출。
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 re?combination. 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 immunogenicity of recombinant spore was measured by ELISA and flowcytometry. 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 spleno?cytes of mice vaccinated with recombinant spores upgraded, which indicated the good humoral and cellular immuni?ties 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.subti?lis 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.