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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 the mage 3 gene, a tumor-specific shared antigen, and prepare an efficient melanoma-specific gene vaccine based on alphavirus replicase. Methods The mage 3 gene was prepared from the melanoma cell line LB3 73 by RT PCR, and the recombinant DNA vaccine was constructed by using pSMART2a, a mammalian cell expression plasmid containing alphavirus replicase. The T7 and T3 promoter sequences in the recombinant vector were sequenced automatically for sequencing primers. The identified recombinant plasmid was then transformed into 293 cells by liposome method, and the expression of mage 3 protein in the transformed cells was identified by immunoblotting and immunohistochemistry. Results The mage 3 pSMART2a recombinant plasmid was constructed correctly, and the expression of mage 3 protein was detected in the 93 cells transformed with this plasmid. Conclusion The recombinant mage 3 pSMART2a plasmid can be used as a tumor-specific DNA vaccine for the next step in the vaccination of tumor animal models and related immunological effects.