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目的构建携带EBV-LMP1Δ基因的DNA疫苗,并与携带LMP1Δ基因的重组非复制5型腺病毒疫苗联合免疫,观察在小鼠体内诱导LMP1Δ特异性细胞免疫应答的效果。方法用PCR方法获得去除癌基因的LMP1Δ基因,插入到pcDNA3.1(+)-His质粒载体中,构建携带LMP1Δ基因的DNA疫苗pcDNA-LMP1Δ。使用pcDNA-LMP1Δ单独或与携带LMP1Δ基因的非复制5型重组腺病毒疫苗Ad-LMP1Δ联合免疫Balb/C小鼠,末次免疫1周后应用IFN-γELISPOT方法检测小鼠脾淋巴细胞中LMP1Δ特异性细胞毒性T淋巴细胞(CTL)水平。结果构建成pcDNA-LMP1ΔDNA疫苗,酶切及测序证实LMP1Δ基因插入正确,在细胞中能有效表达LMP1Δ蛋白。pcDNA-LMP1Δ单独免疫Balb/C小鼠能够诱导产生LMP1Δ特异性的CTL应答;与Ad-LMP1Δ联合使用后,能够提高特异性细胞应答水平。结论构建的重组DNA疫苗pcDNA-LMP1Δ能够有效表达LMP1Δ蛋白,并能够在小鼠体内诱导出LMP1Δ特异性的CTL反应;与重组腺病毒疫苗联合应用,可以提高特异性CTL应答水平。
Objective To construct a DNA vaccine carrying EBV-LMP1Δ gene and immunize it with recombinant non-replicating adenovirus type 5 adenovirus carrying LMP1Δ gene to observe the effect of inducing LMP1Δ-specific cellular immune response in mice. Methods The LMP1Δ gene was amplified by PCR and inserted into pcDNA3.1 (+) - His plasmid vector to construct the DNA vaccine pcDNA-LMP1Δ carrying LMP1Δ gene. Balb / C mice were immunized with pcDNA-LMP1Δ alone or with Ad-LMP1Δ, a non-replicating recombinant adenovirus vaccine carrying the LMP1Δ gene. One week after the last immunization, IFN-γELISPOT method was used to detect LMP1Δ specificity in mouse splenic lymphocytes Cytotoxic T lymphocyte (CTL) levels. Results The pcDNA-LMP1ΔDNA vaccine was constructed. The digestion and sequencing confirmed that the LMP1Δ gene was inserted correctly, and the LMP1Δ protein was efficiently expressed in the cells. Balb / C mice immunized with pcDNA-LMP1Δ alone were able to induce LMP1Δ-specific CTL responses; combined with Ad-LMP1Δ, increased the level of specific cellular responses. Conclusion The constructed recombinant DNA vaccine pcDNA-LMP1Δ can efficiently express LMP1Δ protein and induce LMP1Δ-specific CTL response in mice. Combined with the recombinant adenovirus vaccine, it can enhance the specific CTL response.