基于α-病毒复制酶的高效小鼠P815肿瘤疫苗的构建和表达

目的：克隆小鼠肥大细胞瘤P815细胞株的肿瘤特异PIA基因于含α－病毒复制酶的真核表达载体PSMART中，以制备P815肿瘤DNA疫苗。方法：RT－PCR方法扩增P1A基因，以含α－病毒复制酶的哺乳细胞高效表质质粒PSMART为载体，构建重组肿瘤DNA疫苗，重组体经酶切和测序后，再用脂质体转化293人胚肾细胞，ECL western-blot法和免疫组化法鉴定转化细胞中PIA基因的表达。结果：正确构建了P1A／PSMART重组质粒，并且在转化此质粒的293细胞中检测出了P1A的表达。结论：成功构建了重组P1A／ pSMART肿瘤DNA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 effective vaccine of mouse tumor P815

ve To clone P1A gene from mouse mastocytoma P815 cell strain to mammalian expressing vector containing alpha-virus replicas for producing tumor DNA vaccine. Methods Recombinant DNA vaccine was constructed by ligating P1A gene which was pre-pared by RT-PCR and the vector-pSMART which contains alpha-virus replicase and expresses in mammals with high efficiency. The recombinant was identified with restrictive digestion and sequencing methods, and then was transformed into 293 cells with liposome, the expression of P1 A in 293 cell transformed with the recombinant was checked by ECL western-blot and immunohistochemistry. Results The recombinant P1A/ pSMART plasmid had been constructed correctly and could express the target P1A gene in transformed 293 cells correctly. Conclusion We had constructed successfully the recombinant P1A/pSMART plasmids which could be used as tumor vaccine for inoculatation into tumor animal model at next step and the immunological effect can be determined further.