【摘要】 PCR扩增西门利克森林病毒(SFV)亚基因组启动子26S序列及人工合成Linker,将其插入基于SFV复制子的“自杀性”DNA疫苗表达载体pSCA1的多克隆位点,获得含2个26S启动子并能同时驱动双基因共表达的“自杀性”DNA疫苗表达载体pSCA2。为了验证pSCA2的表达能力,PCR扩增绿色荧光蛋白基因EGFP和红色荧光蛋白基因DsRed2,分别插入pSCA2的2个26s启动子下游,获得EGFP和DsRed2双基因共表达的“自杀性”DNA疫苗pSCA2-EGFP/RFP。将pSCA2-EGFP/RFP转染BHK-21细胞,转染后24h,可同时观察到被转染细胞发绿色荧光和红色荧光,证实EGFP和DsRed2均获得表达。更多还原

【Abstract】 A 0.45 kb DNA fragment containing the 26S promoter of Semliki Forest virus(SFV) and a linker was amplified by PCR and inserted into the multiple clone sites of pSCA1,a suicidal DNA vaccine vector based on SFV replicon,resulting in a modified vector pSCA2 to drive the co-expression of double genes.To further study the expression ability and characterization of the modified expression vector pSCA2,two reporter genes,EGFP and DsRed2,were amplified by PCR and then inserted into the pSCA2,downstream of two 26S promoters respectively,to generate the expression plasmid pSCA2-EGFP/RFP.pSCA2-EGFP/RFP was further transfected into BHK-21 cells.24 h post transfection,green fluorescence and red fluorescence could be observed in all transfected cells using fluorescence microscopy,indicating that the modified expression vector could correctly express two heterologous proteins at the same time.The successful modification of the expression vector pSCA2 offered a useful tool for development of the suicidal DNA vaccine against various pathogens.更多还原