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

作　　者：孙元杰[1] 宋朝君[1] 魏玉英[1] 张贇[1] 金伯泉[1] 杨安钢[1] 杨琨[1] 

机构地区：[1]第四军医大学基础部免疫学教研室,陕西西安710033

出　　处：《第四军医大学学报》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)

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.

关 键 词：BAP31分子 LAMP分子 疫苗 DNA 酶联免疫斑点试验 

分 类 号：R392.11[医药卫生—免疫学]