SQR基因在大肠杆菌和乳酸菌中的表达

Expression of sulfide-quinone reductase(SQR) in Escherichia coli and Lactic acid bacteria

为了研究硫化物-醌氧化还原酶(SQR)基因的乳酸菌表达载体pMG36e-SQR-Myc的构建及其在大肠杆菌DH5a和乳酸菌MG1363中的表达,通过限制性内切酶酶切大肠杆菌-乳酸菌穿梭表达载体pMG36e与目的片段SQR-Myc,回收纯化并用T4连接酶进行连接,连接产物通过KCM法转化到大肠杆菌DH5a中,提取所得到阳性大肠杆菌菌株质粒pMG36e-SQR-Myc进行双酶切鉴定与普通PCR鉴定;并通过电转化将重组质粒导入乳酸菌MG1363中,采用SDS-PAGE电泳和Western-blotting检测SQR蛋白质在其中的表达情况。结果显示,SQR乳酸菌重组表达载体pMG36e-SQR-Myc被成功构建,目的蛋白质SQR在大肠杆菌和乳酸菌MG1363中被成功检测到。因此,乳酸菌MG1363可以用来表达硫化物-醌氧化还原酶(SQR)。

To study the construction of lactococcal expression vector pMG36e-SQR-Myc and its expression in Escherichia coli and Lactococcus lactis MG1363,the expression vector pMG36e and the gene（SQR-Myc） were digested by two restriction endonucleases.SQR-Myc gene was ligated with expression vector pMG36e by T4 DNA ligase,and the ligation products were transferred into Escherichia coli by KCM to get the recombinant vector.The recombinant vector pMG36e-SQR-Myc were verified by double enzyme digestion and PCR amplification.pMG36e-SQR-Myc were transferred into Lactococcus lactis MG1363 by eletroporation.The expression of SQR in Lactococcus lactis MG1363 was detected by SDS-PAGE and Western-blotting.The results showed that the recombinant plasmid pMG36e-SQR-Myc was successfully constructed,and the SQR expression in Escherichia coli and Lactococcus lactis MG1363 were successfully detected.Lactococcus lactis MG1363 could be used for the expression of sulfide-quinone reductase（SQR）.