中华蜜蜂囊状幼虫病病毒单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibodies against Chinese Sacbrood Bee Virus(CSBV)

为了制备中华蜜蜂囊状幼虫病病毒(CSBV)单克隆抗体,本实验利用纯化的中华蜜蜂囊状幼虫病毒(CSBV)免疫Balb/c小鼠,经过3次免疫后,小鼠断尾采血测其血清效价,选择效价高于1∶80 000的小鼠,在细胞融合前3~4d再加强免疫一次。取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,用间接ELISA筛选阳性细胞株并进行3次亚克隆后注射入Balb/c小鼠(Mus musculus)腹腔制备单抗腹水。共获得9株稳定分泌抗CSBV的单克隆抗体,命名为10A1、5B10、5A5、5H2、11D7、9A5、1D3、3C10、10C4,效价都在1∶16 000以上,经间接ELISA实验检测表明,具有较好的特异性。在此基础上,将9株腹水单抗分别与CSBV互作后,接种于2~3日龄中华蜜蜂幼虫,观察幼虫死亡率,研究单克隆抗体中和CSBV病毒能力,结果表明筛选到三株单克隆抗体(10A1、5A5、9A5)对CSBV有中和作用,为CSBV的防治研究奠定了基础。

We wished to prepare and identify monoclonal antibodies against the Chinese honeycomb cystic larvae virus （CSBV）. First, the Chinese sacbrood bee virus was used thrice to immunize Balb/c mice. Then, we collected blood from tail veins to test their serum titer;We selected mice with a titer higher than 1 ： 80,000. We immunized mice again 3-4 days before cell fusion. We undertook cell fusion between the splenocytes of immunized mice and SP2/0 myeloma cells. We applied an indirect enzyme-linked immu- nosorbent assay （ELISA） to screen positive cell strains after cell fusion. After sub-cloning these screened positive cell strains thrice, we injected them into the abdominal cavity of Balb/c mice to prepare ascites monoclonal antibodies. We obtained nine monoclonal antibodies that secreted anti-CSBV steadily and named them 10A1, 5B10, 5A5, 5H2, 11D7, 9A5, 1D3, 3C10 and 10C4. The titers of these nine ascites monoclonal antibodies were higher than 1： 16,000. Results of the indirect ELISA suggested that these nine ascites monoclonal antibodies had good specificity. On the basis of that result, these nine ascites mon- oclonal antibodies were interacted with CSBV, and then inoculated to 2-3-day-old Chinese bee larvae. The mortality of Chinese bee larvae was observed to study the neutralizing capacity of monoclonal antibodies to CSBV. Of the nine ascites monoclonal antibodies screened, three （10A1, 5A5,9A5） had a neutralizing effect no CSBV. The present study lays a foundation for the sdudy of the prevention and treatment of CS- BV.