流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A融合蛋白的表达及其免疫原性研究  

Construction,Expression and Immunogenicity Analysis of a Fusion Protein Containing M2e of Influenza A Virus Fused to a Modified Pseudomonas Aeruginosa Exotoxin A

作　　者：徐一[1] 姚立红[1] 陈爱珺[1] 郭建强[1] 刘晓宇[1] 薄洪[2] 刘丽琦[2] 舒跃龙[2] 张智清[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,国家流感中心,北京100052

出　　处：《病毒学报》2010年第3期189-194,共6页Chinese Journal of Virology

基　　金：传染病防治科技重大专项(编号:2009ZX10004-710)

摘　　要：本研究构建了表达甲型流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A(PEA)融合蛋白的原核表达载体,根据铜绿假单胞菌外毒素A(PEA)核苷酸序列设计突变PCR引物并实施突变PCR,以获得PEA基因编码区第553位氨基酸密码子缺失的突变PEA(ntPE),从而产生无毒性的PEA突变基因,然后用合成的M2e编码区替换ntPE基因中的非必需区Ib,产生ntPE-M2e嵌合基因。将该嵌合基因导入pET表达载体以构建原核表达载体,将表达产物胶回收后与弗氏不完全佐剂联合皮下免疫BALB/c小鼠,终免两周后用5个LD50流感病毒A/PR/34/8株进行攻击。取动物血清作ELISA并取脾脏作ELISPOT试验结果表明,免疫组可以诱导小鼠产生抗M2e特异性抗体反应和细胞免疫反应并能够抑制病毒在肺内的复制。本研究为甲型流感病毒广谱疫苗的进一步研发打下了基础。M2 protein of type A influenza virus is a good candidate for universal influenza vaccine,exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene,and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein （ntPE-M2e） was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund＇s adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein （ntPE-M2e） immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide,and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.

关 键 词：甲型流感病毒M2e 铜绿假单胞菌 外毒素A 免疫原性 

分 类 号：R373.1[医药卫生—病原生物学] R373.9[医药卫生—基础医学]