小鼠肥大细胞瘤P815特异抗原P1A的克隆与表达

目的 克隆小鼠肥大细胞瘤Ｐ８１５细胞株的ＰＩＡ基因以制备肿瘤ＤＮＡ疫苗，方法 用ＲＴ－ＰＣＲ方法制备ＰＩＡ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Ｔ１３启动了序列为测序引物，用自动测序仪测定鉴定克隆的正确性。再将鉴定过的重组质粒用磷酸钙法转化２９３细胞，用ＲＴ－ＰＣＲ法鉴定转化细胞中ＰＩＡ基因的表达。结果 正确构建了ＰＩＡ／ｐＣＩ－ｎ

Cloning and expression of specific antigen P1A of mouse mastocytoma cell line P815

Objective To clone P1A gene from mouse mastocytoma cell strain P815 for the production of tumor DNA vaccine. Methods P1A gene was prepared with RT PCR and then ligated with the vector pCI neo which having high efficient expression in mammals to construct a recombinant DNA vaccine. The recombinant was sequenced by automatic sequencer with promoters T7 in upstream and T3 in downstream as sequencing primers. After the identified recombinant plasmids were transformed into cell 293 with Ca 3(PO 4) 2, the expressions of P1A in the cells were determined with RT PCR.Results The recombinant P1A/pCI neo plasmid was constructed successfully and the expression of P1A gene could be detected in the transformed 293 cells. Conclusion Our experiments indicate that the recombinant P1A/pCI neo tumor DNA vaccine can be constructed successfully which could be used for the studies of tumor animal model.