CAJ | 학술논문

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.Western 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.
목적:구건Sox2급돌변체Sox2 K247R적진핵표체재체, 병재293FT세포중표체.방법:이함유Sox2기인전장적궤증질립위모판, 용순배연신PCR법득도해기인적돌변체Sox2 K247R, 병장야생형급돌변형기인정향아극륭도진핵표체재체pCMV-HA상.득도적중조질립경한제성내절매소화화DNA측서감정.기후용지질체포매법장pCMV-HA-Sox2급pCMV-HA-Sox2 K247R분별단독전염혹여pCMV-Myc-SUMO1공전염293FT세포.채용Western blot분석단백표체정황급SUMO수식정황.결과:경매절화DNA서렬측정, 중조자서렬정학, 돌변체제247위밀마자유AAG전변위CGG.Western blot결과현시야생형급돌변형적Sox2도재세포내득도료흔호적표체, SUMO수식위점돌변후, Sox2불능여SUMO단백결합.결론:Sox2야생형급돌변형진핵표체재체구건성공, 재단백수평체현료SUMO수식적차이성.
AIM:To construct eukaryotic expression plasmids encoding Sox2 and Sox2 K247R and identify their expression and SUMOylation. METHODS: With gift plas-mid encoding Sox2 gene as a template, Sox2 K247R was obtained by overlapping extension PCR, followed by construction of pCMV-HA-Sox2 and pCMV-HA-Sox2 K247R. After enzyme digestion analysis and DNA sequencing, these two constructs were transfected or co-transfected with pC-MV-Myc-SUMO1 into 293 FT cells by lipofectin method. Western blot was employed to analyze expression and SUMO ylation of Sox2. RESULTS: It was revealed that eukaryotic expression vectors were constructed with correct sequence, where in mutant Sox2, the AAG codon was switched to CGG codon. Western blot results showed that good expression of both wt and mut Sox2, of which the latter could not be modified by SUMO1. CONCLUSION: Successful construction and expression of Sox2 and Sox2 K247R. Sox2 could be SUMO lyated in vitro but Sox2 K247R not.