硫化物-醌氧化还原酶(SQR)基因真核表达载体的构建及表达

Construction and expression of eukaryotic vector of sulfide-quinone reductase(SQR) gene

探讨SQR基因真核表达载体pcDNA3.1(-)-SQR-Myc的构建及其在真核细胞中的表达。根据已知荚膜红杆菌SQR基因序列设计5'端和3'端引物,采用PCR方法获得SQR基因,通过pMD-18T载体构建得到中间质粒pMD-18T-SQR-Myc;采用限制性内切酶XhoⅠ和KpnⅠ双酶切该中间质粒,将SQR基因插入真核表达载体pcD-NA3.1(-)相应酶切位点,获得重组质粒pcDNA3.1(-)-SQR-Myc;采用脂质体法瞬时转染HEK293细胞并进行West-ern blot蛋白鉴定。结果显示:该研究已成功构建了真核表达载体pcDNA3.1(-)-SQR-Myc,质粒测序及酶切结果完全正确,并且SQR能够在HEK293细胞系中正常表达。

To construct the eukaryotic vector pcDNA3.1（-）-SQR-Myc and investigate its expression in eukaryocyte,the 5′ primers and 3′ primers were designed and synthesized following the gene sequence of SQR from Rhodobacter capsulatus.The sulfide-quinone reductase（SQR）gene was obtained by PCR and the intermediate vector pMD-18T-SQR-Myc was constructed.The vector was then digested by restriction enzymes XhoⅠand KpnⅠ,and the SQR gene was inserted into the the corresponding restriction enzyme cutting site to construct recombinant plasmid pcDNA3.1（-）-SQR-Myc.The plasmid pcDNA3.1（-）-SQR-Myc was transiently transfected into the HEK293 cells with lipidosome and identified by Western blotting.The results showed that the eukaryotic vector pcDNA3.1（-）-SQR-Myc was constructed successfully,which was confirmed by restriction enzyme digestion and sequencing.Western blotting revealed that SQR could be expressed in HEK293 cells.