异种化PSCA融合基因抗前列腺癌DNA疫苗的构建及真核表达

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（PSCA，）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人真核表达载体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。

Construction and eukaryotic expression of DNA vaccine encoding heterological prostate stem cell antigen for prostate cancer

Objective： To construct a DNA vaccine containing heterological genetic sequence encoding most cytotoxic lymphocyte epitopes of human prostate stem cell antigen（PSCA）--pVAX1-PSCA3-Fc-GPI-IRES-GM/B7 （pVAX1-PSCA3FcGB）, and detect its expression in a eukaryotic cell Cos7. Methods： The heterological PSCA genetic fragment was synthesized by overlapping extending-PCR, and then 3 copies of the fragment （PSCA3） were linked up by co-adhesive end restriction and ligation strategy. The PSCA3 fragment was inserted into a eukaryotic expression vector pCI-Fc-GPI including the gene of the signal peptide of human Igk, human IgG-Fc and glycosyl phosphatidyl inositol（GPI）. At last, the fusion genetic fragment PSCA3-Fc-GPI was cloned into the final eukaryotic expression vector-pVAX1-IRES-GM/B7 which includes several genetic fragments encoding internal ribozyme entry site （IRES）, human grauulocyte-macrophage colony-stinmlating factor （GM-CSF） and costimulatory mole- cules-B7.1 at the downstream of the inserted site. The final recombinant plasmid pVAX1-PSCA3FcGB DNA vaccine was liposomally transfected into Cos7 cells. And the expression of this DNA vaccine was detected by immunofluorescence and flow cytometry. Results ： DNA sequencing result confirmed that the sequence of heterological PSCA genetic fragment was consistent with the design. Enzyme digestion analysis showed that the recombinant plasmid-pVAX1-PSCA3FcGB DNA vaccine was successfully con- structed. The expression of the DNA vaccine in eukaryotic cells was demonstrated by immunofluorescence and flow cytometry. Conclusion： The DNA vaccine -pVAX1-PSCA3FcGB has been successfully constructed and well expressed in Cos7 cells. These results have provided necessary bases for the study of the anti-prostate cancer effect of this vaccine in the future.