Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression.
Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression.
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可溶性原核表达生产生物活性肝靶向干扰素 Mu-IFN-CSP
DOI：
10.1186/s13568-017-0493-z
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发表时间：
2017-10-30
期刊：
影响因子：
3.7
通讯作者：
Lu X
中科院分区：
文献类型：
--
作者：
Liu A;Gui S;Zhang L;Chen Z;Tang Y;Xiao M;Wang J;Liu W;Jin X;Zhu J;Lu X
一种新的肝脏靶向干扰素（IFN-CSP）在我们之前的工作中成功过表达。体外和体内研究表明，IFN-CSP具有显著的抗乙型肝炎病毒（HBV）作用和肝脏靶向能力。然而，由于IFN-CSP倾向于在重组大肠杆菌（E. coli）中形成包涵体，因此高效生产可溶性肝脏靶向干扰素是一个挑战。从生物医学应用的角度来看，有效表达肝脏靶向干扰素并克服其溶解度差的新策略是必要和重要的。本研究以天然IFN-CSP的氨基酸突变体为基础，设计了一种改良的mu-IFN-CSP。同时，通过正交试验优化了大肠杆菌首选密码子mu-IFN-CSP的编码序列，并优化了诱导表达条件。经过氨基酸突变、密码子优化和诱导条件优化，Mu-IFN-CSP在大肠杆菌中的溶解度高达98.4%。结构比较和分子动力学模拟表明，Mu-IFN-CSP形成了三种结构变化，比天然IFN-CSP更稳定。组织切片结合实验显示，Mu-IFN-CSP也能特异性结合肝脏。体外抗hbv活性测定表明，与天然IFN-CSP相比，可溶性Mu-IFN-CSP在HepG2.2.15细胞中具有更强的抗hbv作用。本研究首次报道肝靶向干扰素Mu-IFN-CSP可以可溶性形式表达，也进一步支持其作为肝靶向抗hbv药物的应用。
A novel liver-targeting interferon (IFN-CSP) was successfully over-expressed in our previous work. The in vitro and in vivo investigation revealed that IFN-CSP has significant anti-hepatitis B virus (HBV) effect and liver-targeting capacity. However, due to the IFN-CSP tends to form inclusion bodies in recombinant Escherichia coli (E. coli), efficient production of the soluble liver-targeting interferon is a challenge. In view of biomedical application, novel strategies for efficiently expressing liver-targeting interferon and overcoming its poor solubility are necessary and important. In the present study, a modified mu-IFN-CSP was designed base on the amino acid mutant of the native IFN-CSP. Meanwhile, the coding sequence of mu-IFN-CSP was optimized for E. coli preferred codon and the induction conditions for expression were optimized by an orthogonal test. After amino acid mutant, codon optimization and induction conditions optimization, the solubility of Mu-IFN-CSP in E. coli was up to 98.4%. The structural comparison and molecular dynamic simulation showed that the Mu-IFN-CSP formed three structure changes and were more stable than the native IFN-CSP. Tissue sections binding assays revealed that Mu-IFN-CSP was also able to specific binding to liver. In vitro anti-HBV activity assays showed that the soluble Mu-IFN-CSP has improved anti-HBV effect in HepG2.2.15 cells compared to the native IFN-CSP. The present study reports for the first time that liver-targeting interferon Mu-IFN-CSP can be expressed as soluble form, and also contributes to further support its application as liver-targeting anti-HBV medicine.
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