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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。
OBJECTIVE: To construct and identify two recombinant STAT1α and STAT1β overexpression plasmids (pLJM1-STAT1α and pLJM1-STAT1β) of STAT1 gene by high expression vector pLJM1-EGFP. Methods The cDNA fragment of STAT1α and STAT1β was obtained by PCR using rat liver cDNA as a template. The gene fragment of STAT1α and STAT1β amplified by PCR was cloned into the plasmid pLJM1-EGFP by AgeⅠ and EcoRⅠ restriction enzyme digestion and ligated with T4 DNA ligase The purified vector and the target gene fragment were transformed. The ligation product was transformed into E. coli DH5α competent cells, and the positive clones were selected. The constructed recombinant plasmids were identified by PCR, double enzyme digestion and DNA sequencing. Results PCR and restriction enzyme digestion confirmed that STAT1α and STAT1β gene fragments were successfully inserted into pLJM1-STAT1α and pLJM1-STAT1β recombinant vectors respectively. The results of DNA sequencing showed that the inserted sequences of STAT1α and STAT1β gene were completely correct. Conclusion The pLJM1-STAT1α and pLJM1-STAT1β overexpression recombinant vectors were successfully constructed.