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.coli BL21(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,Western-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.
安玉麟
,
孙瑞芬
,
张鹤龄
,
郭树春
,
闫素丽
. 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[J]. 华北农学报, 2009
, 24(4)
: 84
-87
.
DOI: 10.7668/hbnxb.2009.04.017
:In order to further study expressed level and condition of Glucose oxidase (GOD) from Aspergillus niger 31758 in E. coli,the coding region of GOD from A. niger 3. 758 was inserted into the prokaryotic expression vector pET2 11a,and the recombinant plasmid was transformed into E. coli BL21 (DE3) plysS. The result of SDS2PAGE showed that the GOD gene was expressed by IPTGinduction,and the molecular weight of the fusion protein was about 66. 5 kDa.An2 tiserum was produced in rabbit immunized with commercial GOD,and ELISA analysis proved that the titer of this antibody was 106. The Western2blotting 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.
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