轮状病毒VP4*高聚体的制备及其免疫保护性评价

Polymerization and evaluation of the protective efficacy of rotavirus VP4* proteins

摘要:

在前期工作中发现，截短的轮状病毒VP4*蛋白 (aa 26–476) 在大肠杆菌中能够以可溶形式表达，且在小鼠模型中具有较高的免疫原性和免疫保护性。本研究通过颗粒化进一步提高VP4*蛋白的免疫保护性。通过37 ℃水浴加热处理24 h使VP4*蛋白多聚化，通过高效液相色谱、透射电镜、分析超离等分析VP4*蛋白颗粒化程度，通过酶联免疫吸附试验分析颗粒化对VP4*蛋白与中和抗体反应性的影响；通过差示量热法分析VP4*高聚体的热稳定性；最后，通过小鼠母传抗体模型研究颗粒化对VP4*免疫原性和免疫保护性的影响。结果表明，VP4*蛋白高聚体结构均一，并且相比三聚体，具有更高热稳定性和中和抗体结合活性；在内毒素<20 EU/mg的条件下，与铝佐剂混合，刺激小鼠产生更高滴度的中和抗体；对轮状病毒导致的腹泻具有更高的免疫保护性。综上所述，VP4*高聚体的研究为轮状病毒基因工程亚单位疫苗的研制提供了更广阔的思路。

Abstract:

In previous studies, we found that truncated rotavirus VP4* (aa 26–476) could be expressed in soluble form in Escherichia coli and confer high protection against rotavirus in the mouse mode. In this study, we further improved the immunogenicity of VP4* by polymerization. The purified VP4* was polymerized through incubation at 37 ℃ for 24 h, and then the homogeneity of the particles was analyzed by HPLC, TEM and AUC, while the thermal stability and antigenicity was analyzed by DSC and ELISA, respectively. Finally, the immunogenicity and protective efficacy of the polymers analyzed by a mouse maternal antibody model. The results showed that VP4* aggregated into homogeneous polymers, with high thermostability and neutralizing antibody binding activity. In addition, VP4* polymers (endotoxin <20 EU/dose) stimulated higher neutralizing antibodies and confer higher protection against rotavirus-induced diarrhoea compared with the VP4* trimers when immunized with aluminium adjuvant. In summary, the study in VP4* polymers provides a new strategy for the development of recombinant rotavirus vaccines.