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目的:构建含有人前列腺干细胞抗原(PSCA)主要T细胞表位的DNA疫苗(pVAX1-PSCA3-Fc-GPI-IRES-GM/B7,简称pVAX1-PSCA3FcGB),并在Cos7细胞中表达。方法:重叠延伸PCR合成异种化PSCA基因片段,同尾酶法将该片段3拷贝串联(PSCA3)后插入pCI-Fc-GPI载体中,再将PSCA3-Fc-GPI融合基因片段经酶切后导入真核表达载体pVAX1-IRES-GM/B7中,构建pVAX1-PSCA3FcGB疫苗,并检测其真核表达情况。结果:经测序异种化PSCA片段与设计一致,PCR和酶切鉴定证明pVAX1-PSCA3FcGB构建成功;间接免疫荧光和流式细胞仪检测结果显示,该疫苗在Cos7细胞中获得较好表达。结论:成功构建DNA疫苗pVAX1-PSCA3FcGB,并在Cos7细胞中有效表达,为下一步的DNA疫苗功能研究奠定了重要基础。
OBJECTIVE: To construct a DNA vaccine (pVAX1-PSCA3-Fc-GPI-IRES-GM / B7, abbreviated as pVAX1-PSCA3FcGB) containing the major T cell epitopes of human prostatic stem cell antigen (PSCA) and to express it in Cos7 cells. Methods: The heterologous PSCA gene fragment was amplified by overlap extension PCR. The plasmid was inserted into pCI-Fc-GPI vector after 3 copies of PSCA3 by end-of-line method, and then the PSCA3-Fc-GPI fusion gene fragment was introduced into The eukaryotic expression vector pVAX1-IRES-GM / B7 was used to construct pVAX1-PSCA3FcGB vaccine and its eukaryotic expression was detected. Results: Sequencing of dissimilarized PSCA fragments was consistent with the design. PCR and restriction enzyme digestion proved that pVAX1-PSCA3FcGB was successfully constructed. The results of indirect immunofluorescence and flow cytometry showed that the vaccine was expressed well in Cos7 cells. Conclusion: The DNA vaccine pVAX1-PSCA3FcGB was successfully constructed and expressed efficiently in Cos7 cells, which laid an important foundation for the further study of DNA vaccine function.