摘要
目的 克隆肿瘤特异性共享抗原mage 3基因 ,制备基于α 病毒复制酶的高效黑色素瘤特异性基因疫苗。方法 用RT PCR方法从黑色素瘤细胞系LB3 73中制备mage 3基因 ,以含α 病毒复制酶的哺乳细胞高效表达质粒pSMART2a为载体 ,构建重组DNA疫苗。重组子用载体中的T7和T3启动子序列为测序引物进行自动测序。再将鉴定过的重组质粒用脂质体法转化 2 93细胞 ,用免疫印迹法和免疫组化法鉴定转化细胞中mage 3蛋白的表达。结果 正确构建了mage 3 pSMART2a重组质粒 ,并且在转化此质粒的 2 93细胞中检测出了mage 3蛋白的表达。结论 此重组mage 3 pSMART2a质粒可以作为肿瘤特异的DNA疫苗 ,可进行下一步的肿瘤动物模型的疫苗接种及相关免疫学效应研究。
Objective To clone MAGE 3 gene from melanoma cell strain LB373 to mammalian expressing vector containing alpha virus replicase for preparing tumor specific DNA vaccine. Methods Recombinant DNA vaccine was constructed by ligating RT PCR prepared MAGE 3 gene to the α virus replicase contained vector pSMART2a which could be expressed in mammal cells with high efficiency. The recombinant was sequenced on automatic sequencer with the sequences of T7 and T3 promoters as primers which were in upstream and downstream of multiple cloning site (MCS) respectively. The expression of MAGE-3 in 293 cells transformed with the recombinant by liposome was detected with Western blotting and immunohistochemistry. Results The recombinant plasmid MAGE 3/pSMART2a was constructed correctly and the expression of MAGE 3 gene in the transformed 293 cells was observed. Conclusion The results indicate tumor vaccine containing MAGE 3/pSMART2a is constructed successfully, which can be used for further study on the immune effect of the tumor vaccine in vivo.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2002年第10期1133-1136,共4页
Journal of Third Military Medical University
基金
国家重点基础研究发展规划资助项目 ("973"项目 ) ( 2 0 0 1CB510 0 0 1)
