Construction and expression of eukaryotic vector of sulfide-quinone reductase(SQR) gene
Construction and expression of eukaryotic vector of sulfide-quinone reductase(SQR) gene
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发表时间：
2011
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通讯作者：
L. Quan
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作者：
L. Quan
为构建真核表达载体pcDNA3.1（-）-SQR-Myc并研究其在真核细胞中的表达，根据荚膜红细菌SQR基因序列设计并合成5′和3′引物，通过PCR扩增出硫醌还原酶（SQR）基因，构建中间载体pMD-18 T-SQR-Myc，用XhoⅠ和KpnⅠ双酶切，将SQR基因插入到相应的酶切位点，构建重组质粒pcDNA3.1（-）-SQR-Myc，用脂质体瞬时转染HEK 293细胞，Western blotting鉴定，结果表明，真核表达载体pcDNA3.1（-）-SQR-Myc转染HEK 293细胞后，能稳定表达SQR基因。经酶切和测序鉴定，成功构建了Myc基因，Western blotting结果显示SQR基因在HEK 293细胞中得到了表达。
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.