摘要： 在克隆内蒙古羊源细粒棘球蚴疫苗候选基因Eg95的基础上,构建原核表达载体pET-Eg95并转化E.coli BL21(DE3),优化表达体系,获得Eg95纯化蛋白.将Eg95基因从克隆质粒pMD19-T-Eg95亚克隆到原核表达载体pETa(+)上,构建原核表达载体pET-Eg95,转化E.coli BL21(DE3)诱导表达,优化表达条件.纯化的 Eg95 融合蛋白经 SDS-PAGE和Western Blot鉴定其正确性和免疫学活性.原核表达载体pET-Eg95在大肠杆菌中高效表达,优化的原核表达条件为:当菌液OD600值为0.6时,加入终浓度为0.1 mmol/L的IPTG,37℃,振荡培养5 h.纯化后的蛋白经SDS-PAGE和Western blot鉴定为目的蛋白并具有生物学活性.原核表达载体pET-Eg95构建正确并可高效表达可溶性Nus-Eg95融合蛋白,可作为特异性抗原应用于免疫印迹法检测血清抗体.
关键词:
细粒棘球蚴,
Eg95,
原核表达,
蛋白纯化,
生物活性
Abstract: Constructing prokaryotic expression vector of Eg95 gene from Echinococcus granulosus and expressing fusion protein in E. coli BL21 (DE3) followed by gained purified protein.Eg95 gene was subcloned into pET-44a (+) from clone vector pMD19-T-Eg95 to construct the prokaryotic expression vector pET-Eg95. Nus-Eg95 fusion protein was expressed successfully in E. coli BL21(DE3) at an optimized expression condition and then was purified after identification by SDS-PAGE and western blot. Prokaryotic expression vector pET-Eg95 expressed efficiently in E. coli BL21(DE3) . A high expression level was achieved by inducing the bacteria with 0.1 mmol /L IPTG，at 0.6 of OD600 value for 5 h at 37℃. Purified soluble fusion protein was identified to be the correct target protein by SDS-PAGE and western blot with favorable biological activity. The prokaryotic expression vector pET-Eg95 was constructed successfully and expressed at a high level under the optimized expression condition. The fusion protein can be used to immunoblot assay as a specific antigen react with the antibodies in serum.
Key words:
Echinococcus granulosus,
Eg95,
Prokaryotic expression,
Protein purification,
Biological activity
中图分类号:
李志伟, 王志钢, 湛奎, 陈献威, 杨军, 李洁. 内蒙古羊源细粒棘球蚴Eg95基因原核表达及蛋白鉴定[J]. 华北农学报, 2010, 25(4): 57-60. doi: 10.7668/hbnxb.2010.04.012.
LI Zhi-wei, WANG Zhi-gang, ZHAN Kui, CHEN Xian-wei, YANG Jun, LI Jie. Prokaryotic Expression and Identification of Eg95 Gene from Echinococcus granulosus in Sheep[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(4): 57-60. doi: 10.7668/hbnxb.2010.04.012.