大豆锰超氧化物歧化酶基因在乳酸乳球菌中的分泌表达

Cloning and Expression of Manganese Superoxide Dismutase Gene by Food-Grade Vector in Lactococcus lactis

为使大豆锰超氧化物歧化酶(Mn superoxide dismutase,Mn SOD)在乳酸乳球菌中高效表达,将克隆的Mn SOD基因开放阅读框序列分别重组到质粒p NZ8149和经改造的质粒p NZS上,表达载体p NZ-SOD和分泌表达载体p NZS-SOD,将两者分别以电穿孔法转化乳酸乳球菌L.lactis NZ3900,经Elliker琼脂板筛选、聚合酶链式反应(polymerase chain reaction,PCR)、酶切鉴定正确后,获得的两重组菌株加入Nisin进行诱导表达,对L.lactis NZ3900/p NZ-SOD和L.lactis NZ3900/p NZS-SOD的表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)和酶活性对比分析。结果显示:L.lactis NZ3900/p NZS-SOD菌株表达SOD总活性约是L.lactis NZ3900/p NZ-SOD菌株的1.6倍,是L.lactis NZ3900/p NZ8149的13.5倍,且可将表达的约2/3的SOD分泌到胞外,表明经改造的乳酸菌分泌表达系统L.lactis NZ3900/p NZS能够分泌表达SOD,并增强SOD的表达。

Open reading frame of manganese superoxide dismutase(Mn SOD) from soybean was cloned into the plasmid p NZ8149 and plasmid p NZS was reformed to construct the food-grade vector. The recombinant plasmids were then separately transformed into L. lactis NZ3900 by electronic perforation, and the growth ability of the transformants was detected in Elliker medium. The recombinant plasmids named p NZ-SOD and p NZS-SOD were thus constructed successfully after PCR amplification, ligation and identification. The expression of L. lactis NZ3900/p NZ-SOD and L. lactis NZ3900/ p NZS-SOD was induced by nisin and their expressed products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The SOD enzymatic activities of the transformants L. lactis NZ3900/p NZ-SOD and L. lactis NZ3900/p NZ-SOD were determined by the inhibition of nitrotetrazolium. The SOD enzymatic activity of the transformant L. lactis NZ3900/p NZS-SOD(with two thirds of extracellular SOD) was 1.6 times higher than that of the transformant L. lactis NZ3900/p NZS-SOD and 13.5 times higher than that of the parent strain. Hence, the expression of Mn SOD was enhanced in L. lactis NZ3900/p NZS.