【摘要】 目的构建表达H3、H5、H7、H9亚型流感病毒通用多表位重组鸡痘病毒活载体疫苗候选株。方法筛选流感病毒主要抗原(HANANPM)优势抗原表位基因,以生物信息学软件优化其排列结构合成流感多表位基因(Epi),以H5、H7HA融合表达基因为骨架,将多表位基因插入构建H7HA-Epi-H5HA阅读盒,定向克隆至鸡痘病毒表达载体pUTA-2复合启动子(ATI-P7.5×20)下游,构建重组鸡痘病毒中间转移载体pUTA-2-H7HA-Epi-H5HA。应用脂质体介导的转染法,将该重组鸡痘病毒中间转移载体与282E4株鸡痘病毒共转染鸡胚成纤维细胞(CEF),经BrdU进行3次加压筛选挑斑纯化后传代,并对重组以鸡痘病毒进行PCR和IFA检测和Western blot分析。结果成功构建了重组鸡痘病毒中间转移载体pUTA-2-H7HA-Epi-H5HA,PCR、IFA和Western blot检测阳性。结论筛选出稳定共表达H5H7HA基因和通用多表位基因的重组鸡痘病毒vUTA2-H7HA-Epi-H5HA株,该重组鸡痘病毒的构建为跨种通用型流感病毒活载体疫苗的研究奠定了物质基础。更多还原

【Abstract】 Objective To construct recombinant fowlpox virus (rFPV) co-expressing multi-epitopes of influenza/A subtype H3, H5, H5, H7, and H9 virus. Methods The epitope gene of dominant antigen (HA NA NP M) d of influenza A viruses was selected. The multi-epitopes gene was synthesized and optimized by bioinformatics methods, and then inserted into the H5H7HA gene to construct an ORF, which should be cloned into downstream of the complex prompter (ATI-P7.5×20) of pUTA-2 vector to construct the recombinant fowlpox virus middle transfer vector pUTA-2-H7HA-Epi-H5HA. The CEF was then co-transfected with the reconstructed vector and FPV 282E4 strain under the pressures of Brdu. PCR, IFA, and Western blotting were also utilized to identify the recombinant virus and detect the bioactivity of the expression products in CEF, respectively. Results The middle transfer vector pUTA-2-H7HA-Epi-H5HA for rFPV was constructed successfully. The antibody specificity of the rFPV expressional product was detected by IFA and Western blotting. Conclusion The rFPV obtained by three times of pressure of Brdu could express efficiently, furthermore the expression product can specially recognized by the corresponding antibodies.更多还原