人免疫缺陷病毒包膜蛋白V3多肽与白喉毒素无毒突变体CRM197结构域A融合蛋白的免疫原性分析及单克隆抗体筛选

摘要

Objective To evaluate the potential of CRM197-HIV-V3 fusion protein as an epitope vaccine,to screen monoclonal antibodies and provide guideline for the study of HIV vaccine and therapeutic antibodies.Methods NL4-3-299-328 polypeptide was fused with CRM197 domain A and expressed in E.coli.The 6～8 weeks old Balb/c female mice were immunized with the purified fusion proteins.After 3 times of immunization,following spleen immunization,the immune stimulated spleen cells were hybridized with mouse hybridoma ones.Neutralization monoclonal antibody(NmAb)was screened by neutralization screening platform based on ELISPOT approach.The monoclonal antibody were characterized by HIV neutralization assay and ELISA.ResultsConstruct CRM197-A-V3 was successfully expressed in E.coli,and the resultant fusion protein elicited about 10.3 HIV neutralization titers in mice.The reaction titers of anti-sera was 10.5 as shown in ELISA,and 8 NmAbs were raised.ELISA results showed these NmAbs have good binding activity against NL4-3-gp120 proteins.Neutralization assay showed these NmAbs possess good neutralizing capacity against HIVNL4-3 strain,with NC50 of about 0.02μg/mL.Conclusion The V3 epitope was fused to CRM197 domain A,and significantly evoked the HIV specific immune response in mice.The neutralization monoclonal antibodies against NL4-3 were acquired.%目的 评估CRM197-HIV-V3作为HIV表位疫苗的潜力,以此筛选HIV单克隆中和抗体,为后续疫苗和治疗性抗体的研究奠定基础.方法 将HIV-1 NL4-3 V3肽(299～328 aa)展示在白喉毒素无毒突变体A结构域(CRM197-A)上,利用大肠杆菌原核表达系统进行表达.纯化后的融合蛋白免疫6～8周龄的雌性Balb/c小鼠,免疫3针后,进行脾脏免疫加强,后将小鼠的脾细胞与小鼠杂交瘤细胞进行融合实验.利用基于免疫斑点印迹法(ELISPOT)的HIV中和筛选平台筛选单克隆抗体,对获得的单抗进行抗原性的分析.结果 构建克隆后,成功表达了NL4-3 HIV V3肽融合蛋白(CRM197-A-V3),免疫小鼠的多抗血清对NL4-3株HIV病毒的中和效价约为10.3,ELISA检测多抗血清结合CRM197-A-V3抗原效价达到10.5.通过融合筛选和单细胞克隆化获得 8株单克隆抗体.ELISA结果显示与NL4-3-gp120蛋白有较好的结合活性.中和实验结果显示抗体对NL4-3株HIV病毒有很好的中和能力,NC50为0.02 μg/mL.结论 本研究将HIV V3表位展示在CRM197-A上,成功刺激小鼠产生免疫反应,筛选获得特异性针对NL4-3株HIV的中和单抗.