大豆锰超氧化物歧化酶基因在乳酸乳球菌中的分泌表达

为使大豆锰超氧化物歧化酶（Mn superoxide dismutase，MnSOD）在乳酸乳球菌中高效表达，将克隆的MnSOD基因开放阅读框序列分别重组到质粒pNZ8149和经改造的质粒pNZS上，表达载体pNZ-SOD和分泌表达载体pNZS-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. lactisNZ3900/pNZ-SOD和L. lactis NZ3900/pNZS-SOD的表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodiumdodecyl sulfate - polyacrylamide gel electrophoresis，SDS-PAGE）和酶活性对比分析。结果显示：L. lactis NZ3900/pNZS-SOD菌株表达SOD总活性约是L. lactis NZ3900/pNZ-SOD菌株的1.6 倍，是L. lactis NZ3900/pNZ8149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/pNZS能够分泌表达SOD，并增强SOD的表达。

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

Open reading frame of manganese superoxide dismutase (MnSOD) from soybean was cloned into the plasmid
pNZ8149 and plasmid pNZS 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 pNZ-SOD and pNZS-SOD were thus constructed successfully
after PCR amplification, ligation and identification. The expression of L. lactis NZ3900/pNZ-SOD and L. lactis NZ3900/
pNZS-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/pNZ-SOD and L. lactis
NZ3900/pNZ-SOD were determined by the inhibition of nitrotetrazolium. The SOD enzymatic activity of the transformant
L. lactis NZ3900/pNZS-SOD (with two thirds of extracellular SOD) was 1.6 times higher than that of the transformant
L. lactis NZ3900/pNZS-SOD and 13.5 times higher than that of the parent strain. Hence, the expression of MnSOD was
enhanced in L. lactis NZ3900/pNZS.