NAEK标记HiD-Hin47融合蛋白作为肺炎球菌多糖蛋白结合疫苗载体的研究  

Study on NAEK-labelled HiD-Hin47 fusion protein as a carrier for pneumococcal polysaccharide conjugate vaccine

作　　者：高寒洋 梁何楚 许琳 王浩猛 严志红 史建明 李军强 朱涛[1,2] Gao Hanyang;Liang Hechu;Xu Lin;Wang Haomeng;Yan Zhihong;Shi Jianming;Li Junqiang;Zhu Tao(College of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457,China;Research and Development Center of China CanSino Biologics Inc.,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,300457 [2]天津康希诺生物股份公司研发中心,300457

出　　处：《中华微生物学和免疫学杂志》2021年第11期880-886,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家科技重大专项"重大新药创制"(2019ZX09301106)。

摘　　要：目的研制新型非天然氨基酸标记的HiD-Hin47融合载体蛋白。方法选择融合蛋白氨基酸序列中合适位置的20个氨基酸位点,利用非天然氨基酸标记技术将原有氨基酸位置插入一种含叠氮基的非天然氨基酸N6-(2-叠氮乙氧羰基)-L-赖氨酸(NAEK),对不同位点插入NAEK的融合蛋白进行表达和纯化,纯化后产物经SDS-PAGE分析,观察各位点融合蛋白产量,最终E677位点表达稳定性更高,均能达到野生型的70%,选择E677位点表达的融合蛋白与3型和6B型肺炎球菌多糖发生click反应完成偶联,所得结合物纯化后进行动物实验和免疫学检测,并与CRM197、破伤风类毒素(TT)、HiD以常规结合方式与3型和6B型肺炎球菌多糖结合产物进行对比。结果以HiD-Hin47为载体蛋白的3型肺炎球菌多糖结合物的免疫原性稍优于与其余3组结合物,但差异无统计学意义;以HiD-Hin47为载体蛋白的6B型肺炎球菌多糖结合物的免疫原性显著优于以TT、HiD为载体的结合物,与以CRM197为载体结合物的免疫原性差异无统计学意义。结论NAEK标记的HiD-Hin47具有作为肺炎球菌多糖蛋白结合疫苗载体蛋白的潜力,值得进行进一步研究。Objective To develop an unnatural amino acid-labelled HiD-Hin47 fusion protein as a novel carrier.Methods Twenty versions of the fusion protein were designed,each of which contained a different amino acid site replaced by an azide-bearing amino acid,N6-(2-azidoethoxy)(carbonyl)-L-Lysine(NAEK).These fusion proteins were constructed,expressed and purified,and the yield were evaluated by SDS-PAGE.Based on the highest protein yield,which was approximately 70%of the wild-type yield,the fusion protein with the unnatural amino acid in E677 site was selected.The pneumococcal polysaccharides of serotype 3(F3)and serotype 6B(F6B)were coupled to the selected fusion protein through a"click"reaction.The conjugates were purified and compared in animal studies with other F3 and F6B conjugates that were coupled to CRM197,tetanus toxoid(TT)and HiD by conventional methods.Results The immunogenicity of F3 conjugate using HiD-Hin47 as carrier(F3-HiD-Hin47)was slightly better than that of other F3 conjugates.F6B-HiD-Hin47 was significantly better than F6B-TT and F6B-HiD in terms of immunogenicity,but showed no significant difference with F6B-CRM197.Conclusions NAEK-labelled HiD-Hin47 had the potential as a carrier for pneumococcal polysaccharide conjugate vaccine and was worthy of further study.

关 键 词：融合蛋白 非天然氨基酸 免疫原性 结合疫苗 

分 类 号：R392-33[医药卫生—免疫学]