目的:构建Sox2及突变体Sox2 K247R的真核表达载体,并在293FT细胞中表达。方法:以含有Sox2基因全长的馈赠质粒为模板,用循环延伸PCR法得到该基因的突变体Sox2 K247R,并将野生型及突变型基因定向亚克隆到真核表达载体pCMV-HA上。得到的重组质粒经限制性内切酶消化和DNA测序鉴定。其后用脂质体包埋法将pCMV-HA-Sox2及pCMV-HA-Sox2 K247R分别单独转染或与pCMV-Myc-SUMO1共转染293FT细胞。采用Western blot分析蛋白表达情况及SUMO修饰情况。结果:经酶切和DNA序列测定,重组子序列正确,突变体第247位密码子由AAG转变为CGG。West-ern blot结果显示野生型及突变型的Sox2都在细胞内得到了很好的表达,SUMO修饰位点突变后,Sox2不能与SUMO蛋白结合。结论:Sox2野生型及突变型真核表达载体构建成功,在蛋白水平体现了SUMO修饰的差异性。 Objective: To construct eukaryotic expression vector of Sox2 and Sox2 K247R and express in 293FT cells. Methods: The mutant Sox2 K247R was obtained by cyclic-extension PCR using the plasmid containing the full-length Sox2 gene as a template. The wild-type and mutant genes were subcloned into the eukaryotic expression vector pCMV-HA. The resulting recombinant plasmids were verified by restriction enzyme digestion and DNA sequencing. Thereafter, pCMV-HA-Sox2 and pCMV-HA-Sox2 K247R were transfected individually or co-transfected into 293FT cells with pCMV-Myc-SUMO1 by liposome embedding. Western blot analysis of protein expression and SUMO modification. Results: After digestion and DNA sequencing, the recombinants were correct, and the codon at position 247 of the mutant was changed from AAG to CGG. West-ern blot results showed that the wild-type and mutant Sox2 were well expressed in the cells, Sox2 can not bind SUMO protein after SUMO modification site mutation. Conclusion: Sox2 wild-type and mutant eukaryotic expression vector was successfully constructed, which showed the difference of SUMO modification at the protein level.