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目的:构建上游可以表达肿瘤抗原复合物,下游表达人GM-CSF和B7.1免疫协同增效分子的双顺反子真核表达载体pVAX1-IRES-GM/B7。方法:通过搭桥PCR获得人GM-CSF和B7.1融合基因,插入已构建好的DNA疫苗载体pVAX1-IRES的下游,瞬时转染293T细胞,通过流式细胞术和间接免疫荧光检测融合基因的表达。此外,还在载体上游插入肿瘤抗原复合物,进一步验证该载体对肿瘤抗原的表达情况。结果:酶切鉴定和序列分析表明,GM/B7融合基因与设计完全一致,在体外细胞检测中获得表达,而且上游的抗原基因也可以顺利表达。结论:该载体的构建成功可以为肿瘤基因疫苗研制提供免疫增效载体。
OBJECTIVE: To construct a bicistronic eukaryotic expression vector pVAX1-IRES-GM / B7 that can express tumor antigen complex upstream and downstream and express human GM-CSF and B7.1 immune synergistic molecules. METHODS: Human GM-CSF and B7.1 fusion genes were obtained by PCR. 293T cells were transiently transfected into the downstream of pVAX1-IRES vector. The fusion gene was detected by flow cytometry and indirect immunofluorescence expression. In addition, the tumor antigen complex was also inserted upstream of the vector to further verify the expression of the tumor antigen by the vector. Results: The results of restriction enzyme digestion and sequence analysis showed that the GM / B7 fusion gene was completely identical with the designed one, expressed in the cell culture in vitro, and the upstream antigen gene could be successfully expressed. Conclusion: The successful construction of this vector can provide immuno-synergistic carrier for the development of tumor gene vaccine.