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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.
OBJECTIVE: To enhance the MHC-II presentation of truncated BAP31 tumor antigen (BAP31) and to activate more specific CD4 + T cells through the targeting of LAMP molecules, which ultimately help to increase CTL cell killing activity and specific antibodies Methods: The recombinant plasmids of P-Δ AP31 and PL-Δ AP31 were constructed, and the recombinant plasmids of P-Δ AP31 / EGFP and PL-Δ AP31 / EGFP were constructed. Plasmids P- △ AP31 / EGFP and PL- △ AP31 / EGFP were transient The Hela cells were transfected into Hela cells and the expression of the target protein was observed under a fluorescence microscope. The C57BL / 6 mice were immunized with the recombinant plasmids of P-Δ AP31 and PL-Δ AP31, the specific antibody titers in serum of the immunized mice were detected by indirect ELISA. Lymphocyte secretion of cytokines IFN-γ and IL-4 frequency; lactate dehydrogenase release assay specific CTL response in immunized mice.Results: After digestion and sequence determination, the successful construction of truncated tumor antigen BAP31 DNA Vaccine recombinant vector; transfected Hela cells with recombinant plasmid with EGFP reporter gene, specific green fluorescence was observed by fluorescence microscopy; serum of mice immunized with ELISA showed that the titer of BAP31-specific antibody with LAMP group was significantly higher than that without LAMP group (P <0.01), and both were higher than empty plasmids The frequency of T cells secreting IFN-γ and IL-4 secreted by ELISPOT was significantly higher in LAMP group than in LAMP group (P <0.01). The killing test showed that the specific CTL activity of LAMP group was significantly higher than that of LAMP group (P <0.01) .Conclusion: The constructed P-Δ AP31 and PL-Δ AP31 gene vaccines not only induce specific humoral and cellular immune responses in mice, but also PL- △ AP31 gene vaccine The immune effect was significantly better than that of P-AP31, which lays the foundation for the further development of therapeutic gene vaccines for tumors.