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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。
In this study, a prokaryotic expression vector expressing the extracellular domain of influenza A virus M2 protein and PEA fusion protein was constructed. According to the design of the PEA nucleotide sequence of the Pseudomonas aeruginosa exotoxin A (PEA) The PCR primers were mutated and a mutated PCR was performed to obtain a mutant PEA (ntPE) with deletion of the amino acid codon 553 of the PEA gene coding region to generate a non-toxic PEA mutant gene, which was then replaced with the synthetic M2e coding region Required region Ib, resulting in ntPE-M2e chimeric gene. The chimeric gene was introduced into pET expression vector to construct prokaryotic expression vector, the expression product gel was recovered and BALB / c mice were immunized subcutaneously with incomplete Freund’s adjuvant, and two weeks later, five LD50 influenza A / PR / 34/8 strain. The animal serum was taken for ELISA and the spleen was taken for ELISPOT test. The results showed that the immunized group could induce the mice to produce specific anti-M2e antibodies and cellular immune responses and inhibit the virus replication in the lung. This study laid the foundation for further research and development of a broad-spectrum vaccine against influenza A virus.