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

发布时间：2018-11-09 14:07

【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。
[Abstract]:In this study, a prokaryotic expression vector expressing A (PEA) fusion protein of influenza A virus M2 protein and Pseudomonas aeruginosa exotoxin was constructed. According to the nucleotide sequence of Pseudomonas aeruginosa exotoxin A (PEA), the mutated PCR primers were designed and mutated PCR, was carried out to obtain the mutant PEA (ntPE), of the 553 amino acid codon of the PEA gene to produce the non-toxic PEA mutation gene. The ntPE-M2e chimeric gene was produced by replacing the non-essential region Ib, of the ntPE gene with the synthetic M2e coding region. The chimeric gene was introduced into pET expression vector to construct prokaryotic expression vector. BALB/c mice were immunized subcutaneously with Freund's incomplete adjuvant. Two weeks later, 5 LD50 influenza virus A/PR/34/8 strains were used to attack. The results of ELISA and spleen ELISPOT test showed that the immunized group could induce anti-M2e specific antibody reaction and cellular immune reaction and inhibit the replication of virus in the lung. This study laid a foundation for further research and development of influenza A virus wide-spectrum vaccine.
【作者单位】：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;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;
【基金】：传染病防治科技重大专项(编号:2009ZX10004-710)
【分类号】：R392

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