水痘-带状疱疹病毒糖蛋白gB人源性抗体抗原表位的确定及抗病毒作用研究

Varicella-zoster virus (VZV) is strongly infectious,badly affecting the people who have low immune function. The application of specific immunoglobulins against VZV can effectively avoid or inhibit the process of the disease, and relieve the nerve pain elicited by the virus infection. Passive immune protection plays an important role for the susceptible population who had been exposed to the virus in a certain period of time. Glycoprotein gB of VZV, an envelope protein conserved, has been suggested to play an important role in viral entry and replication. Glycoprotein gB is essential for VZV infection, although cellular receptors for VZV-gB have not been identified and immulogical epitope of gB is not clear. We obtained one EBV-transformed lymphoblastoid cell line (LCL) secreting antibody to VZV-gB in preliminary work. The antibody can identify recombinant and native VZV-gB and can inhibit the virus infection of Vero cells. Active memory B lymphocytes from the patients of herpes zoster in the recovery period are immortalized by EBV transformed technology in this study. Cell lines secreting antibody are chosen and the binding epitope region was analyzed by amino acid point mutation. These findings will pay way for the prevention and treatment of VZV infection.

水痘-带状疱疹病毒（VZV）传染性强，严重威胁免疫力低下人群的健康。针对VZV的特异性免疫球蛋白在暴露于VZV后的短时间内应用，可以有效地避免或减轻水痘进展，并减轻带状疱疹引发的神经疼痛。VZV-gB是水痘-带状疱疹病毒中最为保守的糖蛋白，它在病毒颗粒穿入细胞、病毒包膜形成以及复制过程中发挥重要作用，VZV-gB诱导产生的抗体具有中和作用，但VZV-gB的抗原表位以及对应受体等信息尚不清楚。我们在前期工作中通过EBV转化技术建立一株分泌抗体的淋巴母细胞株，该抗体能识重组和天然的VZV-gB，并能在体外抑制病毒感染Vero细胞。本研究采用EBV转化技术转化带状疱疹恢复期患者记忆性B细胞为淋巴母细胞，筛选抗体阳性的细胞株，通过氨基酸点突变技术确定抗体的抗原结合表位，为设计VZV亚单位疫苗提供实验依据，同时获得的中和性抗VZV-gB抗体为治疗性抗体药物的研究奠定基础。

水痘-带状疱疹病毒（VZV）属于人类疱疹病毒3型，是水痘和带状疱疹两种疾病的病原体。VZV-gB是水痘-带状疱疹病毒中最为保守的糖蛋白，它在病毒颗粒穿入细胞、病毒包膜形成以及复制过程中发挥重要作用。我们通过EBV转化技术建立1株分泌抗体的淋巴母细胞株（LCL），该抗体能识别重组和天然的VZV-gB，并能在体外抑制病毒感染Vero细胞。利用细胞和小鼠模型实验，发现抗体能延迟细胞病变出现的时间，抗体能部分抑制病毒感染小鼠，抗体在病毒感染过程中有免疫保护作用。采用分段构建表达病毒gB蛋白片段的真核载体来分析抗体的结合表位，结果显示C端胞质区的的gB蛋白片段能被抗体抗特异性识别和结合。我们在对稳定生长LCL的选择过程中发现，核型正常的LCL有助于建立稳定的细胞系。此外，我们发现酸性环境对LCL生长特性产生影响。LCL在生长过程中向细胞外分泌大量乳酸，乳酸促使LCL细胞形态学发生改变，细胞粘附能力增强。乳酸促进LCL细胞外基质成分的表达，抑制病毒miR-BHRF1-1的表达，从而使其靶基因（SMAD3、JUN）表达增多，激活TGF-β信号下游分子，促进细胞外基质的表达，促进细胞的侵袭、迁移能力。