黑曲霉葡萄糖氧化酶基因的原核表达及其蛋白产物的Western-blot分析  

Prokaryotic Expression and Western-blot Analysis of Glucose Oxidase Gene from Aspergillus niger

作　　者：安玉麟[1] 孙瑞芬[1] 张鹤龄[2] 郭树春[1] 闫素丽[3] 

机构地区：[1]内蒙古农牧业科学院生物技术中心,内蒙古呼和浩特010031 [2]内蒙古大学生命科学学院,内蒙古呼和浩特010021 [3]内蒙古农业大学农学院,内蒙古呼和浩特010019

出　　处：《华北农学报》2009年第4期84-87,共4页Acta Agriculturae Boreali-Sinica

基　　金：内蒙古自治区优秀学科带头人计划项目(20050802)

摘　　要：为进一步研究黑曲酶中国株3.758葡萄糖氧化酶(GOD)在大肠杆菌中的表达水平和条件,将黑曲霉中国株3.758的葡萄糖氧化酶(GOD)基因定向克隆于原核表达载体pET-11a上,构建了融合表达的重组质粒pET/GO,转化E.coliBL21(DE3)plysS,用IPTG诱导,超声波粉碎细菌细胞,SDS-PAGE电泳检测,GOD基因获得了表达,表达的融合蛋白相对分子质量为66.5 kDa。用市售葡萄糖氧化酶免疫家兔获得抗血清,酶联法(ELISA)测定其效价为106,West-ern-blot分析证明表达的融合蛋白与兔抗GOD血清有较强的特异性,为黑曲霉3.758葡萄糖氧化酶(GOD)抗体的制备奠定了基础。In order to further study expressed level and condition of Glucose oxidase （GOD）from Aspergillus niger 3.758 in E. coli ,the coding region of GOD from A. niger 3.758 was inserted into the prokaryotic expression vector pET- 11a, and the recombinant plasmid was transformed into E. coli BL21 （DE3）plysS. The result of SDS-PAGE showed that the GOD gene was expressed by IPTG induction, and the molecular weight of the fusion protein was about 66.5 kDa. Antiserum was produced in rabbit immunized with commercial GOD, and ELISA analysis proved that the titer of this antibody was 106. The Western-blotting result confirmed that the antibody reacted specifically to the expressed fusion protein. The study established basis for preparation of GOD antibody from Aspergillus niger 3. 758.

关 键 词：黑曲霉3.758 葡萄糖氧化酶基因 融合表达 抗血清 ELISA WESTERN-BLOT 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]