小鼠核糖核酸酶抑制剂异源高效可溶性表达、纯化及活性研究
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摘要
为获得高效可溶性表达的小鼠核糖核酸酶抑制剂(MRNI),本文通过构建含SUMO、IF2、GST、NusA、MsyB、Trx和MBP融合标签的重组表达载体,以大肠杆菌BL21(DE3)作为宿主菌进行自诱导(auto-induction,AI)表达,利用MagNi磁珠检测及电泳分析MRNI的表达状况,并对其培养温度及时间进行优化,同时,与其他公司核糖核酸酶抑制剂(RI)活性进行对比。结果表明,重组菌BL21(DE3)(pNBEII-MRNI)诱导表达的可溶性融合蛋白IF2-MRNI的产量最高,最适诱导条件为37℃,诱导培养6 h,20℃培养24 h。磁珠纯化后获得的融合蛋白IF2-MRNI浓度为3621.3 mg/L,检测其酶活性约为40 U/μL。该酶具有抑制RNase A活性,防止RNA被降解的作用,为RI的生产及应用提供理论基础。
The paper was to obtain efficient soluble expression of mouse ribonuclease inhibitor( MRNI). By constructing a recombinant expression vector containing SUMO,IF2,GST,NusA,MsyB,Trx and MBP fusion tags,E.coli BL21( DE3) was used as a host strain for auto-induction( AI) expression,and MagNi magnetic beads were used for detection.The expression of MRNI was analyzed by electrophoresis,and the culture temperature and time were optimized.The results showed that the soluble fusion protein IF2-MRNI induced by recombinant BL21( DE3)( pNBEII-MRNI) had the highest yield,and the optimal induction condition was 37 ℃,induced for 6 h,and cultured at 20 ℃ for 24 h. The concentration of the fusion protein IF2-MRNI obtained after purification of the magnetic beads was 3621.3 mg/L.Compared with RI activity of other companies,the enzyme activity was about 40 U/μL.The enzyme had the function of inhibiting RNase A activity and preventing degradation of RNA,and provided a theoretical basis for the production and application of RI.
The paper was to obtain efficient soluble expression of mouse ribonuclease inhibitor( MRNI). By constructing a recombinant expression vector containing SUMO,IF2,GST,NusA,MsyB,Trx and MBP fusion tags,E.coli BL21( DE3) was used as a host strain for auto-induction( AI) expression,and MagNi magnetic beads were used for detection.The expression of MRNI was analyzed by electrophoresis,and the culture temperature and time were optimized.The results showed that the soluble fusion protein IF2-MRNI induced by recombinant BL21( DE3)( pNBEII-MRNI) had the highest yield,and the optimal induction condition was 37 ℃,induced for 6 h,and cultured at 20 ℃ for 24 h. The concentration of the fusion protein IF2-MRNI obtained after purification of the magnetic beads was 3621.3 mg/L.Compared with RI activity of other companies,the enzyme activity was about 40 U/μL.The enzyme had the function of inhibiting RNase A activity and preventing degradation of RNA,and provided a theoretical basis for the production and application of RI.
引文
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[15]余小霞,刘晓青,田健,等.来源于枯草芽孢杆菌的漆酶cot A基因克隆与表达及其酶学性质研究[J].中国农业科技导报,2015,17(1):102-108.
[2]Guo W,Cao L,Jia Z,et al.High level soluble production of functional ribonuclease inhibitor in Escherichia coli by fusing it to soluble partners[J]. Protein Expression&Purification,2011,77(2):185-192.
[3]陈可,代娟,甘春扬,等.通过自诱导方法对乙型肝炎病毒聚合酶TP区进行可溶性表达及鉴定[J].中国生物制品学杂志,2017,30(1):25-28,33.
[4]付大伟,陈启蒙,徐伟.融合蛋白Trx-T4 DL可溶性表达、纯化与其生物活性应用[J].食品工业科技,2018,39(7):83-89,96.
[5]Nie Y,Yan W,Xu Y,et al.High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction:Effect of lac operator[J].Plos One,2013,8(10):e78416.
[6]马闪闪,韩来闯,刘亚娟,等.CBM2蛋白的高密度自诱导[J].中国农学通报,2015,31(18):140-145.
[7]Alves N J,Turner K B,Divito K A,et al.Affinity purification of bacterial outer membrane vesicles(OMVs)utilizing a His-tag mutant[J].Research in Microbiology,2017,168(2):139-146.
[8]周李华,叶德萍,王智,等.T4 DNA连接酶酶活力测定方法[J].中国测试,2014,40(2):64-66.
[9]崔倩倩.防治小菜蛾高效绿僵菌工程菌的构建[D].北京:中国农业科学院,2013.
[10]Dammicco S,Goux M,Lemair C,et al. Regiospecific radiolabelling of nanofitin on Ni magnetic beads with[18F]FBEM and in vivo PET studies[J]. Nuclear Medicine&Biology,2017,51:33-39.
[11]杨杰,张筠,孟祥晨.重组蛋白Lux S诱导表达及纯化条件的优化[J].食品科学,2015,36(19):170-175.
[12]Shavrina M S,Zimin A A,Molochkov N V,et al. In vitro study of the antibacterial effect of the bacteriophage T5thermostableendolysin on Escherichia coli cells[J]. Journal of Applied Microbiology,2016,121(5):1282-1290.
[13]付大伟,陈启蒙,孙莹莹,等.T4 DNA聚合酶在大肠杆菌中高效表达、纯化及部分生物学活性分析[J].食品科学,2018,39(16):214-220.
[14]马媛媛,何健民,康永杰.外源性蛋白在大肠杆菌中可溶性表达的策略综述[J].世界科技研究与发展,2015,37(5):627-630.
[15]余小霞,刘晓青,田健,等.来源于枯草芽孢杆菌的漆酶cot A基因克隆与表达及其酶学性质研究[J].中国农业科技导报,2015,17(1):102-108.