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截短型肿瘤抗原BAP31 DNA疫苗的构建及免疫效果分析

Construction of a new DNA vaccine based on truncated BAP31 and study on its anti-tumor effects
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摘要 目的:通过LAMP分子的靶向作用,增强截短型BAP31肿瘤抗原(BAP31)的MHC-II提呈,激活更多的特异性CD4+T细胞,最终辅助增加CTL细胞的杀伤活性及特异性抗体产生.方法:构建重组质粒P-△AP31和P-L-△AP31,同时构建重组质粒P-△AP31/EGFP和P-L-△AP31/EGFP.质粒P-△AP31/EGFP和P-L-△AP31/EGFP瞬时转染Hela细胞,荧光显微镜观察目的蛋白的表达;将重组质粒P-△AP31和P-L-△AP31免疫C57BL/6小鼠,间接ELISA检测免疫小鼠血清中特异性抗体效价;ELISPOT检测免疫脾淋巴细胞分泌细胞因子IFN-γ和IL-4的频率;乳酸脱氢酶释放法检测免疫小鼠特异性CTL反应.结果:经酶切鉴定及序列测定,成功构建截短型肿瘤抗原BAP31的DNA疫苗重组载体;用带EGFP报告基因的重组质粒转染Hela细胞,荧光显微镜观察可见特异性的绿色荧光;ELISA检测免疫小鼠血清,带LAMP组BAP31特异性抗体效价明显高于不带LAMP组(P<0.01),且两者均高于空质粒及正常对照组;ELISPOT检测分泌IFN-γ,IL-4的T细胞频率,带LAMP组明显增高(P<0.01);杀伤试验表明,特异性CTL活性带LAMP组较不带LAMP组显著提高,且均高于对照组(P<0.01).结论:构建的P-△AP31和P-L-△AP31基因疫苗不仅在小鼠体内均可诱导特异性体液和细胞免疫应答,而且P-L-△AP31基因疫苗的免疫效果显著优于P-△AP31,为进一步研制肿瘤治疗性基因疫苗奠定了基础. AIM: To develop an anti-tumor DNA vaccine encoding a truncated protein of murine BAP31(△BAP31 ) to en-hance endogenous antigen trafficking to the MHC-Ⅱ compartment of antigen-presenting cells by LAMP and to study its efficacy to induce cellular and humoral immunity. METHODS: The DNA vaccine was identified by restriction enzyme analysis, sequencing and protein expression. The frequency of cells producing IFN-γ, or IL-4 in splenocytes immunized mice was measured by ELISPOT. Specific antibodies for △BAP31 were determined by indirect ELISA and cytotoxicity of specific CTL was measured by LDH releasing assay. RESULTS: ELISPOT showed that the number of IFN-γ and IL-4-producing ceils in LAMP group was significantly higher than that in control groups. The level of △BAP31 specific antibodies and CTL the cytotoxicity in LAMP group were markedly higher than those in control groups. CONCLUSION: LAMP enhances cellular and humoral response through greater antigen-specific activation of both CD4^+ T cells and CD8^+ T cells.
出处 《第四军医大学学报》 北大核心 2009年第14期1253-1256,共4页 Journal of the Fourth Military Medical University
基金 国家高技术研究发展计划(863计划)重大项目(2006AA02A237) 国家自然科学基金面上项目(30872371)
关键词 BAP31分子 LAMP分子 疫苗 DNA 酶联免疫斑点试验 BAP31 LAMP vaccines, DNA ELISPOT
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