双顺反子真核表达载体pVAX1-IRES的构建与鉴定

Construction and identification of bicistronic eukaryotic expression vector pVAX1-IRES for gene coexpression

目的:为适应恶性肿瘤免疫治疗中多基因联合应用的需要,在真核表达载体pVAX1的基础上,利用内部核糖体进入位点IRES序列,构建双顺反子真核表达载体pVAX1-IRES,以增强DNA疫苗的免疫疗效。方法:通过PCR扩增获得目的基因IRES并定向克隆到pVAX1载体中,然后在IRES序列的上下游分别插入红色荧光蛋白基因DsRed1和增强型绿色荧光蛋白基因EGFP,瞬时转染人胚胎肾细胞293T,通过流式细胞术和免疫荧光验证基因表达。结果:pVAX1-IRES经相应酶切和测序鉴定,与预期设计一致,DsRed1基因和EGFP基因在pVAX1-IRES载体中,不仅可以分别表达,而且可以同时表达,显示该双顺反子真核表达载体构建成功。结论:pVAX1-IRES双顺反子表达载体的构建,为基因联合表达和恶性肿瘤的免疫治疗作了必要准备。

Objective： To construet a bicistronic eukaryotie expression vector to coexpress two independent open reading frames and to satisfy needs of the research on multitarget anti-tumor cancer vaccine, Methods： The IRES gene was amplified and inserted into an cukaryotic expression rector pVAX1 to construct the bicistronic eukaryotic expression vector pVAX1-IRES, Then the DsRedl and EGFP genes were amplified by PCR and inserted into the upper stream and down- stream of IRES gene respectively, The recombinant plasmid pVAX1 -DsRed1-IRES-EGFP was transfected to the 293T cells, and the expression was detected by FACS and immnofluorescenee （IMF）, Results：The enzyme digestion and sequence analysis showed that the hicistronic eukaryotic expression vector pVAX1-IRES was constructed successfully. The expression of recombinant plasmid pVAX1-DsRedl-IRES-EGFP was demonstrated by FACS and IMF. It was shown that the DsRedl and EGFP genes could express in the 293T cells not only separately but also together. Conclusion： These results provide a necessary basis for studying the muhitarget anti-tumor DNA vaccine in the future.