摘要
目的 基于分步偶联策略,借助经典的马来酰亚胺-巯基交联反应与异肽键分子黏合方法(SpyTag/SpyCatcher系统),建立抗原与CpG佐剂的定点共价结合技术。方法 设计合成马来酰亚胺修饰的CpG-1018和N端加有半胱氨酸的SpyTag短肽,将二者于20℃振荡孵育以获得中间产物SpyTag-CpG。将SpyCatcher与蛋白抗原Omp19融合表达,通过亲和层析及凝胶过滤层析获得SpyCatcher-Omp19重组蛋白。通过本实验室构建的志贺菌多糖抗原SpyCatcher-OPS糖蛋白表达系统,获得SpyCatcher-OPS糖蛋白。将SpyTag-CpG分别与SpyCatcher-Omp19重组蛋白和SpyCatcher-OPS糖蛋白连接,获得CpG佐剂与抗原共价偶联终产物CpG-Omp19和CpG-OPS,经SDS-PAGE分离后,通过荧光分析验证终产物结构。结果 马来酰亚胺和FAM同时标记的CpG-1018和SpyTag连接后,SDS-PAGE检测结果可见对应于SpyTag-CpG交联产物的高相对分子质量条带。马来酰亚胺修饰的CpG-1018与SpyTag连接后,质谱分析结果显示,SpyTag-CpG的相对分子质量与其理论相对分子质量相符合。将中间产物SpyTag-CpG与蛋白抗原SpyCatcher-Omp19、多糖抗原SpyCatcher-OPS孵育连接后,考马斯亮蓝染色和FAM荧光检测均可见对应于终产物CpG-Omp19和CpG-OPS的高相对分子质量条带。结论 成功建立基于分步偶联策略的抗原与CpG佐剂定点共价结合技术,获得了CpG佐剂与两种不同类型抗原的偶联终产物CpG-Omp19和CpG-OPS,为未来开发质量可控的自佐剂疫苗奠定了基础。
Objective To establish a site-specific covalent binding technology of antigens and CpG adjuvants.Methods Maleimide-modified CpG-1018 and N-terminal cysteine-modified SpyTag peptide were designed and synthesized.They were incubated at 20℃ with shaking to obtain the intermediate product SpyTag-CpG. The SpyCatcher was fused and expressed with the protein antigen Omp19,and the target SpyCatcher-Omp19 recombinant protein was obtained by affinity chromatography and gel filtration chromatography. The SpyCatcher-OPS glycoprotein was obtained through the Shigella polysaccharide antigen SpyCatcher-OPS glycoprotein expression system constructed in our laboratory. Subsequently,SpyTag-CpG was incubated with SpyCatcher-Omp19 recombinant protein and SpyCatcher-OPS recombinant glycoprotein respectively to obtain the final products CpG-Omp19 and CpG-OPS,which were the product of antigens covalently coupled with CpG adjuvants. The structure of the final products was verifed by fluorescence analysis after SDS-PAGE.Results After ligation of CpG-1018 labeled with maleimide and FAM simultaneously with SpyTag,a higher relative molecular mass band corresponding to the SpyTag-CpG cross-linked product was seen in SDS-PAGE. After the maleimidemodified CpG-1018 was linked to SpyTag,mass spectrometry analysis showed that the relative molecular mass of SpyTag-CpG was consistent with its theoretical relative molecular mass. After the intermediate product SpyTag-CpG was incubated with the protein antigen SpyCatcher-Omp19 and the polysaccharide antigen SpyCatcher-OPS,higher relative molecular mass bands corresponding to the final products CpG-Omp19 and CpG-OPS were seen by Coomassie Brilliant Blue staining and fluorescence detection. Conclusion A site-specific covalent binding technology of antigens and CpG adjuvants based on the step-by-step coupling strategy has been established,and the final products CpG-Omp19 and CpG-OPS,which are conjugated between CpG adjuvants and two different types of antigens,are obtained.
作者
孙燕歌
黄竞
石怡昕
郭艳
潘超
王恒樑
朱力
SUN Yan-ge;HUANG Jing;SHI Yi-xin;GUO Yan;PAN Chao;WANG Heng-liang;ZHU Li(State Key Laboratory of Pathogen and Biosecurity,Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)
出处
《军事医学》
CAS
CSCD
2023年第2期127-134,共8页
Military Medical Sciences
基金
国家自然科学基金(U20A20361)。
