MAGE-3 DNA疫苗的构建及其免疫效果的观察

通过RT PCR方法扩增MAGE 3cDNA ,以pcDNA3 1+为载体 ,构建重组表达质粒pcDNA3 1 MAGE 3。重组质粒用脂质体转染鼠B16细胞 ,经RT PCR、细胞免疫染色及免疫印迹法鉴定转化细胞中MAGE 3的表达。以 10 0 μg质粒剂量肌肉注射接种小鼠 ,间隔 10天 ,共 3次 ,以空载体和PBS为对照。结果 ,重组质粒免疫的小鼠 ,其脾淋巴细胞对MAGE 3阳性靶细胞的杀伤活性为 51 0 8± 7 41% ,与空载体组 (8 44± 1 89% )及PBS组 (5 76± 1 75% )相比 ,差异有显著性 (P <0 0 1) ,而对MAGE 3阴性靶细胞的杀伤活性分别为 8 2 1± 1 65% ,7 68± 1 56%和 5 13±1 42 % ,其差异无显著性 ;MAGE 3DNA疫苗组免疫血清 1∶15稀释时能检测到抗MAGE 3抗体 ,脾细胞培养上清中Th1类细胞因子IFN γ、IL 2水平明显升高 ,外周血中CD4+、CD8+T细胞也提高 ,小鼠肿瘤的生长速度明显减慢 ,与对照组相比 ,差异显著 (P <0 0 1)。说明MAGE 3重组质粒免疫小鼠可以诱导小鼠产生特异性的体液和细胞免疫应答

Construction of Human MAGE-3 DNA Vaccine and Its Immune Effects Observed in vivo

To investigate the antitumor immune responses induced by MAGE-3 DNA vaccine, the recombinant mammalian expression plasmid pcDNA3.1/MAGE-3 was constructed by ligating MAGE-3 gene, which was amplified by RT-PCR, and the pcD-NA3.1 + vector. The recombinant plasmids were transfected into B16 cells by liposome, the expression of MAGE-3 was checked by RT-PCR, immunocytochemistry and Western blot. Then, 100 ug recombinant plasmids were injected intramuscularly per C57BL/6 mouse on 0, 10 and 20 days, with pcDNA3.1 + plasmid and PBS as controls. Splenocytes CTLs, the level of antibodies against MAGE-3 the changes of the T lymphocyte subsets and the levels of cytokines were checked after 3 times immunization. As a result, the mice immunized with pcDNA3.1/MAGE-3 plasmid can produce MAGE-3 specific immune response. The CTLs kill activities against B16/MAGE-3 cells was 51.08 +/- 7.41%, and had significant difference (P < 0.01) compared with that of pcDNA3.1 + group (8.44 +/- 1.89%) and PBS group (5.76 +/- 1.75%). The titre of antibody against MAGE-3 was 1:15, while controls were negtive. The number of CD4 + CD8 + and the levels of IFN-gamma IL-2 increased significantly after immunization with pcDNA3.1/MAGE-3 plasmid as compared with those of control groups (P < 0.01). It is concluded that the pcDNA3.1-MAGE-3 DNA vaccine are able to induce both cellular and humoral immune responses in vivo.