禽流感病毒M2e、NP多表位嵌合肽抗原的构建及免疫原性

Construction and immunogenicity of chimeric peptide antigen composed of M2e and NP genes of avian influenza virus

摘要:

摘要: 【目的】为了克服传统禽流感疫苗各亚型之间无交叉保护的缺陷，研制抗禽流感通用型疫苗。【方法】 以禽流感病毒M2e、NP表位为基础串联T细胞表位构建4个原核表达载体: pET-3M2e、pET-3M2e-NP1,2-Fc、pET-3M2e-NP1,2、pET-TCE-3M2e-NP1,2。纯化重组蛋白并与弗氏佐剂混合制成疫苗，胸部肌肉注射免疫20日龄非免鸡(150μg/只)，4个融合蛋白分为四组，每组十只。ELISA方法检测血清中M2e抗体水平；在MDCK细胞上检测血清与H9N2亚型禽流感病毒的结合能力，在鸡胚上检测其中和能力；以流式细胞仪测定CD4+、CD8+T淋巴细胞的变化。【结果】研究发现，各免疫组均能检测到高水平的ELISA抗体，免疫荧光显示抗血清均能跟病毒特异性结合，中和试验表明抗血清不能中和病毒但能抑制病毒的复制。流式细胞仪检测显示外周血CD4+、CD8+T淋巴细胞所占比例在免疫后均有明显升高(P<0.05)，TCE-3M2e-NP1, 2组CD4+、CD8+T淋巴细胞所占比例分别达到47.23%和36.77%，具有细胞免疫的特征。【结论】构建的嵌合肽抗原具有较好的免疫原性，能刺激机体产生体液和细胞免疫，为进一步研制抗禽流感通用型疫苗做出有益的探索。

Abstract:

Abstract: [Objective] In order to overcome the defect of traditional avian influenza vaccine that lacks cross-protection among different serotypes, we developed a universal anti-influenza vaccine. [Methods] Based on the gene analysis for AIV matrix protein 2 and two cytotoxic T-lymphocyte epitopes, we constructed four prokaryotic expression vectors. The target gene was induced by IPTG and the fusion protein was mixed with Freund’s adjuvant; then used to immunize20-day-old chicken by intramuscular injection and boosted 3 weeks later. Blood samples were collected weekly following the primary vaccination. The anti-M2e antibody was detected with ELISA coated by synthesized peptide; the neutralizing ability of anti-serum was evaluated on MDCK cell line and chick embryo; the CD4+ and CD8+ T lymphocyte amounts in peripheral blood of immunized chicken were measured by flow cytometry. [Results] The fusion protein induced immunological reaction, and the antibody bound with the viral M2 protein expressed on the surface of MDCK cells. Serum could only inhibit the replication without neutralizing the virus. Flow cytometry results showed that CD4+ and CD8+ T lymphocyte in peripheral blood increased obviously following immunization (P<0.05), which possessed the character of cell immunization. [Conclution] Chimeric peptides kept good immuno-genicity and provided useful probes for the control of avian influenza.