人巨细胞病毒gH蛋白表达及其单克隆抗体的制备

人巨细胞病毒(HCMV)是全球范围内普遍感染的一种疱疹病毒,也是引起胎儿先天性畸形和器官移植受者死亡最常见的病原体。有研究表明,gH蛋白是HCMV主要的中和性抗原,特异性抗gH抗体具有中和HCMV及阻断病毒在细胞间传播的潜力。本研究以含人巨细胞病毒临床株Toledo全基因组的细菌人工染色体(BAC)为模板,扩增去除信号肽和跨膜区的gH基因片段,并将其插入表达载体构建pET32a’-gH重组表达质粒,将序列鉴定正确的重组质粒转化进入表达菌株TransB,诱导重组蛋白表达。用纯化的重组gH蛋白免疫8周龄BALB/c雄鼠,经杂交瘤技术获得5株稳定分泌抗gH单克隆抗体的细胞株,分别命名为8A9、8B4、8C4、8D9、8D12。所获5株单克隆抗体对gH蛋白均具有良好的反应性,其中8A9、8B4、8D9和8D12具有一定的病毒捕获能力,且8D9和8D12的捕获能力较强。本研究获得了具有自主知识产权的抗gH单克隆抗体,抗体具有良好的病毒捕获能力。在此基础上,我们将进一步鉴定其是否为中和抗体,为今后开发治疗HCMV感染的中和抗体奠定基础。

Expression of Human Cytomegalovirus gH Protein and Preparation of Its Monoclonal Antibody

Human cytomegalovirus(HCMV)is a kind of herpesvirus which is widely infected all over the world.It is also the most common pathogen that causes fetal congenital malformations and death of organ transplant recipients.Some studies showed that gH protein was the main neutralizing antigen of HCMV,and specific anti-gH antibodys had the potential to neutralize HCMV and block virus transmission between cells.In this study,the gH gene fragment without signal peptide and transmembrane region was amplified,whose template was the BAC containing the whole genome of human cytomegalovirus clinical strain Toledo,and inserted into the expression vector to construct the recombinant expression plasmid pET32a′-gH.The recombinant plasmid with correct sequence identified was transformed into the expression strain TransB,and then TransB was induced to express the recombinant protein.Eight-week-old male BALB/c mice were immunized with purified recombinant gH protein.Five cell lines stably secreting anti-gH monoclonal antibodies were obtained by hybridoma technique,which were named 8A9,8B4,8C4,8D9 and 8D12 respectively.All the five monoclonal antibodies had good reactivity to gH protein.Among them,8A9,8B4,8D9 and 8D12 had certain virus capture ability,and the capture ability of 8D9 and 8D12 was stronger.In this study,we obtained anti-gH monoclonal antibodies with independent intellectual property rights,and the antibodies had good virus capture ability.On this basis,we will further identify whether they are neutralizing antibodys,which will lay a foundation for the development of neutralizing antibodies for the treatment of HCMV infection in the future.