摘要
乙肝病毒核心蛋白(hepatitis B virus core protein,HBc)可在体外自组装成二十面体的病毒样颗粒(virus-like particles,VLPs),但是其较低的自组装效率及稳定性制约其应用.通过蛋白质改造修饰,在野生型HBc(wtHBc)的免疫显性区(major immunodominant region,MIR)分别插入带电多肽序列RRRRRRRR和DDDDDDDD,以通过荷电基团及静电相互作用的引入提高VLPs稳定性.经大肠杆菌重组表达,成功获得携带额外电荷的HBc突变体(mHBc),包括携带额外正电荷的m+HBc和负电荷的m-HBc.结果表明,mHBc的二级结构富含α-螺旋,与wtHBc一致.m+HBc和m-HBc等比例混合可在体外实现自组装,所得mHBc VLPs粒径为(30.44±2.23)nm,与野生型一致.该自组装过程及组装体稳定性受离子强度和pH值影响.使用尺寸排阻色谱、原子力显微镜、动态光散射等方法表征和分析VLPs在不同缓冲液中的稳定性差异.发现wtHBc VLPs可稳定存在于0.3~0.7 mol/L NaCl溶液中,盐浓度过高或过低均会使其稳定性降低.pH响应性实验表明,wtHBc VLPs的最佳组装及稳定pH值为7.4,偏离此pH值使其稳定性降低.在低于pH=5.5的环境下,其相对稳定性明显降低,只达到pH=7.4的64.2%.而mHBc VLPs的稳定性差异随pH值变化不大,在pH测试范围5.5~9.5的相对稳定性均高于88.3%.以上研究结果表明,正、负电荷基团的引入可提高HBc VLPs对pH值变化的耐受性,拓宽稳定pH值区间,增强VLPs的稳定性,利于其实际应用.
The hepatitis B virus core protein(HBc)can self-assemble into icosahedral virus-like particles(VLPs).However,their application is limited by their low assembly efficiency and low stability.For this paper,charged polypeptides RRRRRRRR or DDDDDDDD were inserted into the major immunodominant region(MIR)of wild-type HBc(wtHBc)to improve VLPs stability through the electrostatic interactions formed by the charged groups.The mutants(mHBc)were then obtained by expression in recombinant escherichia coli,including one form with an extra positive charge(m+HBc)and one with a negative charge(m-HBc).Anα-helix structure was observed in mHBc,which was consistent with wtHBc.Using the equimolecular mixtures of m+HBc and m-HBc,successful selfassembly into VLPs nanoparticles with a particle size of(30.44±2.23)nm was observed,which was consistent with wtHBc VLPs.Assembly process and stability were affected by ionic strength and pH values.The change in stability of the VLPs in different buffers was characterized by size exclusion chromatography,atomic force microscopy,and dynamic light scattering.It was found that the wtHBc VLPs could exist in solutions containing 0.3—0.7 mol/L NaCl.Reduced stability was observed once their ionic strength deviated from this range.Optimized assembly of wtHBc VLPs was obtained at pH=7.4.Deviation from this pH value caused a decrease of stability.Significant decrease of stability was observed when pH value fell below 5.5,which was only 64.2%of that at pH=7.4.For mHBc VLPs,no obvious difference in stability was observed at various pH values,and relative stability of more than 88.3%was observed at the tested pH range of 5.5—9.5.Thus,the improved tolerance of HBc VLPs to pH changes could be obtained by the introduction of extra electrostatic interactions through charged amino acid residues;and,thus,a broader stable pH range and enhanced stability of VLPs were achieved,which could be beneficial to its applications.
作者
张麟
陈衡
Zhang Lin;Chen Heng(School of Chemical Engineering and Technology,Tianjin University,Tianjin 300072,China)
出处
《天津大学学报（自然科学与工程技术版）》
EI
CSCD
北大核心
2020年第5期450-458,共9页
Journal of Tianjin University：Science and Technology
基金
国家自然科学基金资助项目(21978205,91534119)
国家重点研发计划资助项目(2018YFA0900700)
天津大学自主创新基金项目.
关键词
乙肝病毒核心蛋白
电荷
病毒样颗粒
纳米结构
蛋白质修饰
hepatitis B virus core protein(HBc)
charge
virus-like particles(VLPs)
nanostructure
protein modification
