摘要
旨在制备柯浩体的标志蛋白——Atcoilin蛋白,利用pET-28a与目的基因构建重组表达质粒,经DNA测序证实插入序列与设计完全一致后,将重组质粒转化大肠杆菌BL21(DE3),用IPTG进行诱导表达,产物用SDS-PAGE及Western blotting分析鉴定。通过分别改变IPTG的浓度、培养时间、培养温度等来优化Atcoilin蛋白的表达条件。表达出的重组蛋白经过镍柱、分子筛进行纯化。结果显示,原核表达载体pET28a-At1g13030成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相应的重组蛋白经Western blotting鉴定正确。在IPTG浓度为0.7 mmol/L,18℃培养20 h的条件下,目的蛋白表达量最高。经过SDS-PAGE分析鉴定,过镍柱、分子筛后得到的重组蛋白纯度较高。
For the sake of a high expression of Atcoilin,which is a symbol of Cajal bodies（CBs）,the recombinant vector pET28a-At1g13030 was constructed by pET-28a and the target gene.The recombinant protein was induced by IPTG in E.coli BL21（DE3）and the expressed protein was detected by SDS-PAGE and Westernblotting.Through changing the concentration of IPTG,length of time induced by IPTG and culture temperature to optimize Atcoilin expression.Results indicated that the recombinant vector pET28a-At1g13030 was successfully constructed and the Atcoilin protein could be expressed in E.coli BL21（DE3）.The recombinant protein was characterized by Western blotting. Through optimizing,the satisfactory expression condition was obtained,as follow including IPTG concentration 0.7 mmol/L;induced time of 20 h;culture temperature 18℃.The protein obtained through the nickel column and molecular sieve was identified by SDS-PAGE.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第3期63-68,共6页
Biotechnology Bulletin
基金
国家自然科学基金项目(31071075)
