\r张 麟，陈 衡\r
\r
AuthorsHTML:\r张 麟，陈 衡\r
\r
AuthorsListE:\rZhang Lin，Chen Heng\r
\r
AuthorsHTMLE:\rZhang Lin，Chen Heng\r
\r
Unit:\r天津大学化工学院，天津 300072\r
\r
Unit_EngLish:\rSchool of Chemical Engineering and Technology，Tianjin University，Tianjin 300072，China\r
\r
Abstract_Chinese:\r\r乙肝病毒核心蛋白\r(\rhepatitis B virus core protein\r，\rHBc\r)\r可在体外自组装成二十面体的病毒样颗粒\r(\rvirus\r-\rlike particles\r，\rVLPs\r)\r，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\rHBc\r(\rwtHBc\r)\r的免疫显性区\r(\rmajor immunodominant region\r，\rMIR\r)\r分别插入带电多肽序列\rRRRRRRRR\r和\rDDDDDDDD\r，以通过荷电基团及静电相互作用的引入提高\rVLPs\r稳定性．经大肠杆菌重组表达，成功获得携带额外电荷的\rHBc\r突变体\r(\rmHBc\r)\r，包括携带额外正电荷的\rm\r＋\rHBc\r和负电荷的\rm\r-\rHBc\r．结果表明，\rmHBc\r的二级结构富含\rα\r-\r螺旋，与\rwtHBc\r一致．\rm\r＋\rHBc\r和\rm\r-\rHBc\r等比例混合可在体外实现自组装，所得\rmHBc VLPs\r粒径为\r(\r30.44\r±\r2.23\r)\rnm\r，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\rpH\r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\rVLPs\r在不同缓冲液中的稳定性差异．发现\rwtHBc VLPs\r可稳定存在于\r0.3\r～\r0.7mol/L NaCl\r溶液中，盐浓度过高或过低均会使其稳定性降低．\rpH\r响应性实验表明，\rwtHBc VLPs\r的最佳组装及稳定\rpH\r值为\r7.4\r，偏离此\rpH\r值使其稳定性降低．在低于\rpH\r＝\r5.5\r的环境下，其相对稳定性明显降低，只达到\rpH\r＝\r7.4\r的\r64.2\r%\r．而\rmHBc VLPs\r的稳定性差异随\rpH\r值变化不大，在\rpH\r测试范围\r5.5\r～\r9.5\r的相对稳定性均高于\r88.3\r%\r．以上研究结果表明，正、负电荷基团的引入可提高\rHBc VLPs\r对\rpH\r值变化的耐受性，拓宽稳定\rpH\r值区间，增强\rVLPs\r的稳定性，利于其实际应用．\r\r
\r
Abstract_English:\r \r\r\rThe 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\r-\rHBc). An α-helix structure was observed in mHBc, which was consistent with wtHBc. Using the equimolecular mixtures of m\r+\rHBc and m\r-\rHBc, successful self-assembly into VLPs nanoparticles with a particle size of \r(\r30.44\r±\r2.23\r)\r 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\r—\r0.7 mol/L NaCl. Reduced stability was observed once their ionic strength deviated from this range. Optimized assembly of wtHBc VLPs was obtained at pH\r＝\r7.4. 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\r＝\r7.4. 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\r—\r9.5. 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.\r\r
\r
\r
Keyword_Chinese:乙肝病毒核心蛋白；电荷；病毒样颗粒；纳米结构；蛋白质修饰\r

Keywords_English:hepatitis B virus core protein(HBc)；charge；virus-like particles(VLPs)；nanostructure；protein modification\r


PDF全文下载地址:http://xbzrb.tju.edu.cn/#/digest?ArticleID=6448