摘要
目的:构建一系列含有人前列腺干细胞抗原(PSCA)主要T细胞表位的多拷贝异种化融合基因片段,并分别在人胚肾293T细胞中表达。方法:通过重叠延伸PCR法合成单拷贝异种化PSCA基因片段PSCA1,随即应用同尾酶法将该片段串联形成2、3、4拷贝异种化PSCA基因片段PSCA2、PSCA3和PSCA4,并将上述4种基因片段分别插入真核表达载体pCI-Fc-GPI中,构建最终目的片段1～4拷贝异种化PSCA-Fc-GPI(即PSCA1-Fc-GPI～PSCA4-Fc-GPI),随即分别将重组质粒pCI-PSCA1-Fc-GPI～PSCA4-Fc-GPI体外转染293T细胞,利用间接免疫荧光和流式细胞仪检测其表达情况。结果:测序证实PSCA1片段与设计一致,酶切鉴定证明目的基因片段PSCA1-Fc-GPI～PSCA4-Fc-GPI构建成功;间接免疫荧光和流式细胞仪的检测结果显示,在293T细胞中1～4拷贝异种化PSCA融合基因片段均获得较好表达。结论:构建了目的基因片段PSCA1-Fc-GPI～PSCA4-Fc-GPI,为以PSCA为靶抗原的抗前列腺癌DNA疫苗的构建及功能研究奠定了重要基础。
Objective： To construct a series of fusion genetic fragments mainly containing multi-copies heterological genetic sequence which encoding most cytotoxic lymphocyte epitopes of human prostate stem antigen（PSCA）, and detect their expression in eukaryotic cell 293T. Methods： Single copy of heterological PSCA genetic fragment（PSCA 1） was constructed by overlapping-extending-PCR, and the co-adhesive end restriction and ligation strategy was used to link the former fragment one by one. Then the 1 to 4 copies fragments PSCA I-PSCA4 were inserted into eukaryotic expression vector pCI-Fc- GPI respectively, to construct the final aimed genetic fragments, PSCA1-Fc-GPI-PSCA4-Fc-GPI. Four kinds of corresponding recombinant plasmids were transfected into 293T cells, and the expression of four kinds of fusion genetic fragments were detected by immunofluorescence and flow cytometry. Results： DNA sequencing conformed that the construction of PSCA1 was consistent to design. Enzyme digestion analysis showed that the final aimed genetic fragments of PSCA1-Fc- GPI-PSCA4-Fc-GPI were constructed successfully. And corresponding recombinant plasmids expressed well in 293-T cells. Conclusion： We have constructed PSCA1-Fc-GPI-PSCA4-Fc-GPI fusion genetic fragments successfully. These results have provided necessary bases for constructing anti-prostate cancer DNA vaccine targeting PSCA in the future.
出处
《生物技术通讯》
CAS
2009年第2期147-150,共4页
Letters in Biotechnology
基金
国家自然科学基金(30772002)
国家高技术研究发展计划(2006AA02A237
2007AA02Z451)
