摘要
目的对青海血蜱重组r Hq002蛋白包涵体进行复性及纯化。方法将重组质粒pET-30a-Hq002转化E. coli Rosetta(DE3),IPTG诱导表达重组Hq002蛋白;超声破碎菌体,收集细胞碎片,用PBS缓冲液和低浓度尿素进行多次洗涤;洗涤后的包涵体用6 mol/L盐酸胍溶解,稀释法进行蛋白复性;用镍离子亲和层析法纯化蛋白后,分别于4和-20℃保存7 d后进行12%SDS-PAGE鉴定。结果经PBS缓冲液和低浓度尿素多次洗涤,可去除大量的水溶性杂质和部分水不溶性杂蛋白,初步纯化了包涵体;包涵体经6 mol/L盐酸胍溶解后可直接稀释复性重组蛋白;经镍离子亲和层析纯化后的蛋白相对分子质量约27 000,与预期相符,4和-20℃保存7 d未出现降解或者聚集。结论成功获得含二硫键的蜱源重组蛋白,本研究的复性纯化方法可为纯化包涵体蛋白提供参考。
Objective To re-naturalize and purify recombinant Hq002 inclusion body of Haemaphysalis qinghaiensis.Methods Recombinant plasmid pET-30 a-Hq002 was transformed to E. coli Rosetta(DE3) and induced with IPTG to express r Hq002. The bacterial cells were subjected to ultrasonication,of which the debris were collected and washed for several times with PBS buffer and low concentration urea. The inclusion bodies after washing was dissolved with 6 mol/L guanidine hydrochloride,re-naturalized by dilution,purified by nickel ion affinity chromatography,stored at 4 and-20 ℃for 7 d respectively,and identified by 12% SDS-PAGE. Results Inclusion bodies were preliminarily purified by removing most of water-soluble and partial water-insoluble impurities by washing with PBS buffer and low concentration urea for several times. The re-naturalized r Hq002 was diluted directly after dissolution of inclusion bodies with 6 mol/L guanidine hydrochloride. The relative molecular mass of protein purified by nickel ion affinity chromato-graphy was about27 000,which was consistent with that expected. No degradation or aggregation was observed in the protein after storage at 4 and-20 ℃ for 7 d. Conclusion Tick-derived recombinant protein containing disulfide bond was successfully obtained. The methods for renaturation and purification provided a reference for purification of inclusion body protein.
作者
马静
陈开廷
曹美娜
刘玉婷
马婧
高金亮
MA Jing;CHEN Kai-ting;CAO Mei-na;LIU Yu-ting;MA Jing;GAO Jin-liang(Baotou Medical College,Batou 014000,Inner Mongolia Autonomous Region,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第1期91-94,99,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金项目(81760375)
内蒙古自然科学基金项目(2021MS08063)
内蒙古医科大学“科技百万工程”项目(YKD2020KJBW)。
关键词
蜱源蛋白质
包涵体
复性
纯化
Tick-derived protein
Inclusion body
Renaturation
Purification
