结核分枝杆菌新型亚单位疫苗的初步研究  

Preliminary study on a novel subunit vaccine of Mycobacterium tuberculosis

作　　者：时宇 陶玉芬[1] 刘建生[1] 刘红旗[1] SHI Yu;TAO Yu-fen;LIU Jian-sheng;LIU Hong-qi(Infection and Immunity Laboratory,Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,China)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所感染和免疫实验室,云南昆明650118

出　　处：《云南大学学报（自然科学版）》2020年第4期768-774,共7页Journal of Yunnan University(Natural Sciences Edition)

基　　金：中国医学科学院医学与健康科技创新工程重大协同创新项目(2016-12M-3-028,2017-12M-2-006).

摘　　要：为了研发结核新型亚单位疫苗,以纳米颗粒为载体展示结核分枝杆菌主要抗原ESAT6.将ESAT6基因片段克隆到位于pGEX-4T-1载体上的诺如病毒P结构域的Loop2中,转入大肠杆菌BL21(DE3)进行表达,用GST亲和柱纯化,并通过FPLC分析嵌合纳米颗粒的形成效率,最后免疫BALB/c小鼠进行免疫原性的评价.结果表明:研究成功地将ESAT6基因克隆至诺如病毒P结构域的Loop2中,且空间结构模拟提示ESAT6能展示在P纳米颗粒的表面.FPLC分析表明,表达的嵌合蛋白可以高效地自我组装成纳米颗粒.用该嵌合颗粒免疫小鼠后,能诱导产生针对ESAT6重组嵌合颗粒的特异性抗体.进一步分析发现,诱导的抗体主要针对ESAT6的51~70肽段,而不是1~20肽段.综上,研究通过诺如病毒P颗粒成功地展示了结核分枝杆菌主要抗原ESAT6,为进一步研发结核新型亚单位疫苗奠定了基础.To develop a novel subunit vaccine for Mycobacterium tuberculosis(Mtb),biological nanoparticle was used to display the main antigen of Mtb,ESAT6.The gene fragment of ESAT6 was cloned into the loop2 of the norovirus P domain in pGEX-4T-1.The resulting chimeric gene was expressed in E.coli BL21(DE3),and the chimeric protein was purified by the GST affinity column.The formation efficiency of chimeric nanoparticle was analyzed by the fast protein liquid chromatography(FPLC),and the immunogenicity was evaluated by immunizing BALB/c mice.The results showed that we successfully cloned ESAT6 gene into the Loop2 of norovirus P domain.Protein structure homology-model showed that ESAT6 could be displayed on the surface of P nanoparticle.FPLC analysis showed that the purified chimeric protein could self-assemble into nanoparticle efficiently.Immunization of mice with this chimeric nanoparticle induced specific antibody against ESAT6 nanoparticle.Further analysis revealed that the induced antibody was mainly against the peptide 51-70aa of ESAT6,rather than the peptide 1-20aa.In conclusion,the main antigen ESAT6 of Mtb was successfully displayed by norovirus P particle in this study,which laid a foundation for the further development of novel Mtb subunit vaccine.

关 键 词：结核 ESAT6 纳米颗粒 亚单位疫苗 中和抗原表位 

分 类 号：R378.911[医药卫生—病原生物学]