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目的:构建重组表达载体P-BAP31和P-L-BAP31基因疫苗并接种小鼠,评价其诱导的体液和细胞免疫应答。方法:利用分子克隆技术,构建重组质粒P-BAP31和P-L-BAP31,同时构建P-BAP31/EGFP和P-L-BAP31/EGFP重组质粒,并将其以脂质体瞬时转染HeLa细胞,通过荧光显微镜观察目的蛋白的表达;将重组质粒P-BAP31和P-L-BAP31免疫C57BL/6小鼠,采用间接ELISA检测免疫小鼠血清中特异性抗体效价;通过酶联免疫斑点实验(ELISPOT)检测免疫脾细胞在受到BAP31抗原刺激后产生IFN-γ和IL-4细胞因子的频率;通过乳酸脱氢酶(LDH释放)试验检测小鼠特异性细胞毒性T淋巴细胞(CTL)的应答。结果:酶切及测序结果表明,成功构建了针对BAP31肿瘤抗原的P-BAP31和P-L-BAP31基因疫苗。通过荧光显微镜观察重组质粒转染的HeLa细胞,可见EGFP报告基因表达的绿色荧光。ELISA检测免疫小鼠血清中特异性抗体效价发现,带LAMP组明显高于不带LAMP组(P<0.05),且两者均高于空质粒(P-LAMP)及正常对照(N)组(P<0.01)。ELISPOT检测脾细胞分泌细胞因子IFN-γ和IL-4的频率,带LAMP组与其他组相比显著提高(P<0.01)。LDH释放试验显示,带LAMP组的特异性CTL活性高于不带LAMP组,且均高于对照组(P<0.01)。结论:构建了针对肿瘤抗原BAP31的全长基因疫苗,以其免疫C57BL/6小鼠可诱导特异性体液和细胞免疫反应,其中,P-L-BAP31基因疫苗各项免疫反应均优于P-BAP31。
Objective: To construct recombinant P-BAP31 and P-L-BAP31 gene vaccines and inoculate mice to evaluate their humoral and cellular immune responses. Methods: The recombinant plasmids P-BAP31 and PL-BAP31 were constructed by molecular cloning. The recombinant plasmids of P-BAP31 / EGFP and PL-BAP31 / EGFP were constructed and transiently transfected into HeLa cells by lipofectamine. The recombinant protein P-BAP31 and PL-BAP31 were used to immunize C57BL / 6 mice. The specific antibody titers in the serum of the immunized mice were detected by indirect ELISA. The immune spleen cells were detected by ELISPOT The frequency of IFN-γ and IL-4 cytokines produced by cells stimulated with BAP31 antigen; the response of mouse-specific cytotoxic T lymphocytes (CTL) was tested by lactate dehydrogenase (LDH release) assay. Results: The results of enzyme digestion and sequencing showed that P-BAP31 and P-L-BAP31 gene vaccines against BAP31 tumor antigen were successfully constructed. HeLa cells transfected with the recombinant plasmids were observed by fluorescence microscope, and green fluorescence of EGFP reporter gene was observed. The titer of specific antibody in the serum of mice immunized with LAMP was higher than that without LAMP (P <0.05), and both of them were higher than those of P-LAMP and N (P <0.01). The frequency of cytokine IFN-γ and IL-4 secreted by spleen cells was detected by ELISPOT. Compared with other groups, LAMP group was significantly increased (P <0.01). The LDH release test showed that the specific CTL activity of LAMP group was higher than that of LAMP group (all P <0.01). CONCLUSION: The full-length gene vaccine targeting tumor antigen BAP31 was constructed, which can induce specific humoral and cellular immune responses in C57BL / 6 mice. Among them, the immune responses of P-L-BAP31 vaccine were better than that of P-BAP31.