人黑色素瘤抗原MAGE—3基因的克隆与表达

目的 克隆人黑色素瘤特异抗原MAGE-3基因，以制备肿瘤DNA疫苗。方法 用RT-PCR方法制备MAGE-3基因，以哺乳细胞高效表达质粒pCI-neo为载体，构建重组DNA疫苗。重组体用载体上的通用引物为测序引物，鉴定克隆的正确性。再将鉴定过的重组质粒用脂质体法转化293细胞，用免疫印迹法鉴定转化细胞中MAGE-3基因的表达。结果 正确构建了MAGE-3/pCI-neo重组质粒，并且在转化细胞中检测出了MAGE-3的表达。结论 成功地构建了重组MAGE-3/pCI-neo肿瘤核酸疫苗，可以进行下一步的肿瘤动物模型的疫苗接种及疗效观察。

Cloning and expressing of human melanoma antigen MAGE-3

ObjectiveTo clone human melanoma antigen MAGE 3 gene to produce tumor DNA vaccine Methods Recombinant DNA vaccine was constructed by ligating MAGE 3 gene, which was prepared by RT PCR and the vector pCI neo that can express in mammals with high efficiency The recombinant was sequenced on an automatic sequencer with T7 promoter and T3 promoter sequence as sequencing primers that locate upstream and downstream of the multiple cloning site (MCS) respectively and the correctness of recombinant was determined The identified recombinant plasmids were then transformed into 293 cells with liposome, the expression of MAGE 3 was checked by western blot ResultsThe recombinant MAGE 3/pCI neo plasmid had been constructed correctly and the expression of MAGE 3 gene in transformed 293 cells had been determined ConclusionWe had constructed successfully the recombinant MAGE 3/pCI neo tumor vaccine Its anti tumor effects could be observed further in animal models