【摘要】 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更多还原

【Abstract】 To investigate the antitumor immune responses induced by MAGE-3 DNA vaccine, th e recombinant mammalian expression plasmid pcDNA3 1/MAGE-3 was constructed by ligating MAGE-3 gene, which was amplified by RT-PCR, and the pcDNA3 1+ vector . The recombinant plasmids were transfected into B16 cells by liposome, the expr ession of MAGE-3 was checked by RT-PCR, immunocytochemistry and Western blot . Then, 100ug recombinant plasmids were injected intramuscularly per C57BL/6 mou se on 0, 10 and 20 days, with pcDNA3 1+ plasmid and PBS as controls. Splenocyte s CTLs、 the level of antibodies against MAGE-3、the changes of the T lymphocyt e 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 t hat of pcDNA3 1+ group(8 44±1 89%) and PBS group(5 76±1 75%). The titre of an tibody against MAGE-3 was 1∶15, while controls were negtive. The number of CD 4+、CD8+ and the levels of IFN-γ、IL-2 increased significantly after immuniz ation 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 i nduce both cellular and humoral immune responses in vivo.更多还原