猪细小病毒中和性单克隆抗体的制备与鉴定

Preparation and Identification of Neutralizing Monoclonal Antibodies against Porcine Parvovirus

为了制备具有中和活性的抗猪细小病毒(Porcine parvovirus,PPV)单克隆抗体,采用血凝试验(HA)鉴定纯化的重组PPV VP2蛋白的活性,将重组VP2蛋白与弗氏佐剂混合后免疫BALB/c小鼠。免疫3次后,小鼠血清的血凝抑制(HI)效价可达1∶2^(16),取免疫小鼠脾细胞与SP2/0细胞融合制备杂交瘤细胞,采用有限稀释法进行多轮亚克隆,经免疫过氧化物酶单层细胞试验(IPMA)筛选,成功获得杂交瘤细胞株5F7和11B3,能够稳定分泌中和性单克隆抗体。单克隆抗体5F7和11B3的轻链型均为Kappa,重链型分别为IgG2a和IgG2b。经ELISA和IPMA检测,单克隆抗体5F7和11B3均能与重组PPV VP2蛋白和PPV病毒粒子发生特异反应,而与猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)无交叉反应。5F7和11B3腹水针对PPV病毒反应的ELSIA效价分别为1∶10 240和1∶20 480;针对PPV感染PK15细胞的中和效价分别为1∶2^(11)和1∶2^(10)。Western blot鉴定结果显示,单克隆抗体5F7和11B3均不与变性的VP2蛋白发生反应,说明2株单克隆抗体均识别重组PPV VP2蛋白的构象型表位。综上,成功制备了2株具有中和活性的抗PPV单克隆抗体。

In order to prepare neutralizing monoclonal antibody against porcine parvovirus(PPV),the recombinant PPV VP2 protein was purified and its hemagglutination(HA)activity was detected.BALB/c mice were immunized with the recombinant protein mixed with Freund’s adjuvant.After three immunizations,the serum hemagglutination inhibition(HI)titer of mice was up to 1∶2^(16).The spleen cells of immunized mice were fused with SP2/0 cells.After subcloning and immunoperoxidase monolayer assay(IPMA)screening,two hybrioma cell lines,5F7 and 11B3,which could secrete neutralizing monoclonal antibody stably were successfully obtained.The light chains of the antibodies were Kappa type,the heavy chain of 5F7 was IgG2a subtype,11B3 was IgG2b.Monoclonal antibodies 5F7 and 11B3 reacted specifically with recombinant PPV VP2 protein and PPV virions by ELISA and IPMA,but did not reac with porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV)and classical swine fever virus(CSFV).The titers of monoclonal antibodies 5F7 and 11B3 neutralizing PPV to infect PK15 cells were 1∶2^(11) and 1∶2^(10),respectively.The ELSIA titers of monoclonal antibodies 5F7and 11B3 reacting with PPV virus were 1∶10 240 and 1∶20 480.Western blot showed that monoclonal antibodies 5F7 and 11B3 did not react with denatured VP2 protein,indicating that both monoclonal antibodies recognized the conformational epitopes of recombinant PPV VP2 protein.In conclusion,two monoclonal antibodies with neutralizing PPV infection activity were successfully prepared.