摘要
目的 开发针对新冠病毒BA.5变异株的高免疫原性抗原,并筛选出具有高效中和阻断活性的单克隆抗体,为疫苗和诊断试剂的开发提供新策略。方法 将斑马鱼卵黄蛋白中的ZP蛋白基因片段和HIS蛋白标签插入p CDNA3.4质粒,构建pCDNA3.4-RBD(BA.5)-ZP-HIS重组载体,并转入293F细胞表达RBD-ZP蛋白。通过SDS-PAGE等方法验证蛋白表达,并检测其与ACE2受体分子和铝佐剂的结合能力。利用BALB/c小鼠模型评估融合蛋白的免疫原性,并通过杂交瘤技术制备单克隆抗体。筛选出具有强中和阻断活性的单克隆抗体,并通过阻断ELISA方法检测抗体的中和活性。结果 ZP基因和HIS蛋白标签序列插入到pCDNA3.4载体中,并成功表达出50 kDa的RBD-ZP蛋白。免疫原性检测结果表明,ZP蛋白有效增强了RBD蛋白的免疫原性,并提高了其与ACE2受体的结合能力。进一步用RBD-ZP蛋白对小鼠进行免疫,通过杂交瘤技术克隆、筛选得到了8种能够与突变株和野生株特异性结合的单克隆抗体,其中3种能够有效阻断新冠病毒RBD蛋白与人体ACE2受体的结合。结论 本研究成功表达了RBD-ZP融合蛋白,显著增强了RBD蛋白的免疫原性和受体结合能力,并筛选出3种具有高效中和阻断活性的单克隆抗体,为新冠疫苗和诊断试剂的开发提供了有力支持。
Objective To develop high-immunogenicity antigens targeting the BA.5 variant of SARS-CoV-2 and to screen for monoclonal antibodies with high neutralizing and blocking activity,providing new strategies for the development of vaccines and diagnostic reagents.Methods The ZP protein gene fragment from zebrafish vitel-logenin and the HIS protein tag were inserted into the pCDNA3.4 plasmid to construct the recombinant plasmid pCDNA3.4-RBD(BA.5)-ZP-HIS,which was then transfected into 293F cells to express the RBD-ZP protein.The expression of the protein was verified by SDS-PAGE and its binding capabilities to ACE2 receptor molecules and aluminum adjuvant were detected.The immunogenicity of the fusion protein was evaluated using a BALB/c mouse model,and monoclonal antibodies were prepared through hybridoma technology.Monoclonal antibodies with strong neutralizing and blocking activity were screened and their neutralizing activity was detected by blocking ELISA.Results The ZP gene and HIS protein tag sequence were successfully inserted into the pCDNA3.4 vector and the RBD-ZP protein with a molecular weight of 50 kDa was successfully expressed.The immunogenicity test results showed that the ZP protein effectively enhanced the immunogenicity of the RBD protein and improved its binding capability to the ACE2 receptor.After immunizing mice with the RBD-ZP protein,8 monoclonal antibodies that specifically bind to both the mutant and wild-type strains were cloned and screened through hybridoma technology,among which 3 could effectively block the binding of the SARS-CoV-2 RBD protein to the human ACE2 receptor.Conclusion This study successfully expressed the RBD-ZP fusion protein,which significantly enhanced the immunogenicity and receptor binding capability of the RBD protein.Three monoclonal antibodies with high neutralizing and blocking activity were screened out,providing strong support for the development of COVID-19 vaccines and diagnostic reagents.
作者
吴凡
覃鸿妮
谢钰珍
任保永
戴东升
WU Fan;QIN Hongni;XIE Yuzhen;REN Baoyong;DAI Dongsheng(Suzhou Industrial Park Institute Of Services Outsourcing,Suzhou 215000,Jiangsu,China;不详)
出处
《实用医学杂志》
北大核心
2025年第17期2653-2660,共8页
The Journal of Practical Medicine
基金
国家自然科学基金资助项目(编号:22077092)
江苏省高等学校优秀科技创新团队
江苏省高校“青蓝工程”项目
江苏省精准诊疗药物创制工程研究中心开放课题(编号:SDGC2239)。
作者简介
通信作者:覃鸿妮,E-mail:qinhn@siso.edu.cn。
