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[目的]构建人PSCA和PAP融合基因的真核表达质粒,并在体外进行表达和鉴定。[方法]设计并合成人PSCA和PAP融合基因,然后将其定向克隆到真核表达载体p IRES-neo中,双酶切鉴定后,将重组质粒p IRES-neoPSCA-2A-PAP瞬时转染293T细胞,采用流式细胞术和免疫荧光细胞化学技术验证其表达。[结果]构建了p IRES-neo-PSCA-2A-PAP真核表达质粒,流式细胞术和免疫荧光细胞化学技术结果显示融合抗原可以在293T细胞表达,阳性率高达67.85%。[结论]成功构建了融合抗原PSCA与PAP的真核表达质粒p IRES-neo-PSCA-2A-PAP,为建立稳定转染人PSCA和PAP融合基因的细胞系奠定了基础。
[Objective] To construct eukaryotic expression plasmid of human PSCA and PAP fusion gene and to express and identify in vitro. [Method] The fusion gene of human PSCA and PAP was designed and synthesized, and then cloned into the eukaryotic expression vector p IRES-neo. After double enzyme digestion, the recombinant plasmid pESES-neoPSCA-2A-PAP was transiently transfected into 293T The cells were verified by flow cytometry and immunofluorescence cytochemistry. [Results] The eukaryotic expression plasmid pIRES-neo-PSCA-2A-PAP was constructed. The results of flow cytometry and immunofluorescence cytochemistry showed that the fusion antigen was expressed in 293T cells with a positive rate of 67.85%. [Conclusion] The eukaryotic expression plasmid pIRES-neo-PSCA-2A-PAP fused with the PSCA and PAP plasmids was successfully constructed and laid the foundation for establishing a cell line stably transfected with human PSCA and PAP fusion genes.