【摘要】 【目的】探讨以流感病毒(IAV)血凝素蛋白茎部(HA2)保守表位为免疫原来诱导针对不同血清型流感病毒的广谱体液免疫应答。【方法】我们构建了一个重组的IAV疫苗IR30-Fd,由A/Viet Nam/1203/2004(H5N1)病毒株HA2的30个保守的氨基酸残基(IR30)和一个连在碳端的三聚体基序(foldon,Fd)组成。原核表达该蛋白,圆二色谱和Western Blot验证IR-30-Fd的室间结构之后,分别免疫BALB/c小鼠和新西兰白兔,并同时免疫IR30多肽为对照,制备血清抗体。然后应用ELISA和Western Blot方法检测抗体的滴度,应用细胞ELISA和Western Blot方法检测抗体与不同的IAV菌株HA2蛋白的交叉反应性。【结果】IR30-Fd能够形成具有α螺旋的三聚体空间结构。用IR30-Fd和IR30免疫BALB/c小鼠和新西兰白兔后获得了高效价的抗IR30的特异性抗体。鼠抗IR30-Fd抗体能与H3N2和H1N1的HA蛋白反应而鼠抗IR30抗体不能。鼠抗IR30-Fd抗体稀释6 400倍、兔抗IR30-Fd抗体稀释8.2×105倍均可以与H5N1的HA蛋白反应。细胞ELISA表明兔抗IR30-Fd抗体与天然状态下的H5N1和H3N2的HA蛋白的结合亲和力高于兔抗IR30抗体。【结论】本研究结果表明IR30-Fd蛋白能够模拟H5N1 HA2蛋白保守序列的天然三聚体结构,并且能够诱导产生针对H5N1 H1N1和H3N2亚型HA蛋白的广谱交叉反应抗体,为研制通用的流感病毒疫苗提供了结构基础。更多还原

【Abstract】 【Objective】 This study aimed to find epitopes in HA2 that can induce broadly cross-reactive antibody responses against divergent influenza A viruses(IAVs). 【Methods】 We constructed a recombinant IAV vaccine containing a 30 amino acid conserved sequence(IR30) from HA2 of the IAV strain A / Viet Nam / 1203 / 2004(H5N1) plus a trimeric motif(foldon), designated as IR30-Fd, which was expressed in Escherichia coli(E.coli) using prokaryotic fusion protein expression system. The secondary structures of IR30-Fd and IR30 were analyzed by circular dichroism spectroscopy and Western blot. We immunized mice and rabbits with IR30-Fd protein or IR30 peptide(as a control), and detected antibody titers of the sera using ELISA and Western Blot. We then employed cell ELISA and Western blot to determine the cross-reactivity of antibodies against divergent IAV strains. 【Results】 IR30-Fd can form an α-helical trimer.We found that both anti-IR30-Fd and anti-IR30 antibodies were highly effective in binding to IR30. However, mouse anti-IR30-Fd antibodies could interact with HA of H3N2 and H1N1, while anti-IR30 antibodies could not. Mouse anti-IR30-Fd antiserum at the dilution as high as 1:6,400 could react with HA of H5N1. Rabbit anti-IR30-Fd antiserum at the dilution as high as1:8.2x105 could react with HA of H5N1. Furthermore, rabbit anti-IR30-Fd antibodies showed significantly higher affinity than antiIR30 antibodies to the native conformation of HA of H5N1and H3N2 expressed on Hela cells. 【Conclusion】 These results suggest that IR30-Fd, which could mimic the native trimeric form of the conserved sequence in HA2 region of H5N1, was able to induce broadly cross-reactive antibody against HA of H5N1, H1N1, and H3N2 subtypes, thus serving as a basic structure for developing universal influenza vaccines.更多还原