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目的 克隆小鼠肥大细胞瘤P815细胞株的P1A基因以制备肿瘤DNA疫苗。方法 用RT PCR方法制备P1A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。重组体用克隆位点上游和下游的T7和T3启动子序列为测序引物 ,用自动测序仪测序鉴定克隆的正确性。再将鉴定过的重组质粒用磷酸钙法转化 2 93细胞 ,用RT PCR法鉴定转化细胞中P1A基因的表达。结果 正确构建了P1A/pCI neo重组质粒 ,并且在转化此质粒的 2 93细胞中检测出了P1A的表达。结论 成功地构建了重组P1A/pCI neo肿瘤疫苗 ,可以进行下一步的肿瘤动物模型的疫苗接种及疗效观察
Objective To clone the P1A gene of mouse mastocytoma P815 cell line for preparation of tumor DNA vaccine. Methods The P1A gene was prepared by RT PCR and pCI neo plasmid for mammalian cells was used as a vector to construct a recombinant DNA vaccine. The T7 and T3 promoter sequences upstream and downstream of the cloning site for the recombinant were used as sequencing primers, and the correctness of the clone was verified by sequencing using an automated sequencer. The identified recombinant plasmid was then transformed into 293 cells by the calcium phosphate method, and the expression of the P1A gene in the transformed cells was identified by RT PCR. Results The P1A/pCI neo recombinant plasmid was constructed correctly, and the expression of P1A was detected in the 93 cells transformed with this plasmid. Conclusion The recombinant P1A/pCI neo tumor vaccine was successfully constructed, which can be used for the next step in the vaccination of tumor animal models and the efficacy observation