【摘要】 目的利用高表达载体pLJM1-EGFP构建并鉴定转录因子STAT1基因的2个亚型STAT1α和STAT1β的过表达重组质粒(简称为pLJM1-STAT1α和pLJM1-STAT1β)。方法以大鼠肝脏cDNA为模板,PCR获取目的片段(即STAT1α和STAT1β);用AgeⅠ和EcoRⅠ双酶切pLJM1-EGFP表达载体和PCR扩增的STAT1α和STAT1β基因片段后,用T4DNA连接酶连接酶切纯化后的载体及目的基因片段;将连接产物转化DH5α大肠杆菌感受态细胞、挑选阳性克隆,并通过PCR、双酶切和DNA测序鉴定构建的重组质粒。结果 PCR和双酶切证实pLJM1-STAT1α和pLJM1-STAT1β重组载体中分别成功插入了STAT1α和STAT1β基因片段。DNA测序结果表明插入的STAT1α和STAT1β基因片段的序列完全正确。结论成功构建了pLJM1-STAT1α和pLJM1-STAT1β高表达重组载体。更多还原

【Abstract】 Objective To construct and identify the over-expression vector of transcription factor STAT1α and STAT1β(pLJM1-STAT1α and pLJM1-STAT1β)using pLJM1-EGFP expression vector.Methods The target fragments,STAT 1α and STAT 1β,were obtained by PCR using E3 rat liver cDNA as template.The target fragments and pLJM1-EGFP vector were cut by AgeI and EcoRI restriction endonucleases and then purified enzyme-digested products were ligated together with a T4 DNA ligase.Subsequently,the ligation productions were used to transform DH5α competent cells,and the positive clones were picked up from the agarose plate with ampicillin.Finally,the recombinant plasmids were identified through PCR,double-restrict-enzyme digestion of AgeI and EcoRI and DNA sequencing.Results PCR and double-restrict-enzyme digestion of AgeI and EcoRI confirmed that the target fragments were successfully inserted into the recombinant vectors of pLJM1-STAT1α and pLJM1-STAT1β,respectively.The analysis of DNA sequencing further confirmed that pLJM1-STAT1α and pLJM1-STAT1β successfully inserted the target fragment of STAT1α and STAT1β.Conclusion We successfully constructed the over-expression plasmid of STAT1α and STAT1β,pLJM1-STAT1α and pLJM1-STAT1β.更多还原