H3N2亚型SIV血凝蛋白单克隆抗体的制备

Preparation of monoclonal antibody for H3N2 subtype swine influenza virus hemagglutinin protein

【目的】制备H3N2亚型猪流感病毒(SIV)的单克隆抗体,为SIV的鉴别诊断奠定基础。【方法】将A/swine/Henan/1/2010(H3N2)猪流感病毒初步浓缩后,免疫6周龄的BALB/c小鼠,取小鼠脾细胞与瘤细胞NS0融合,采用间接ELISA方法筛选杂交瘤细胞,对杂交瘤细胞分泌的单克隆抗体进行Western blot检验和抗原结合活性检测。【结果】经过3次有限稀释法克隆纯化,最终得到1株能稳定分泌单克隆抗体的阳性细胞株,命名为1C10,经检测其腹水抗体效价为1∶51 200。单克隆抗体亚型鉴定结果表明,1C10为IgG1亚型,轻链类型为κ链。Western blot检测结果表明,这株单克隆抗体能与H3N2特异性结合,而且能特异性识别血凝素蛋白(HA),而不与H1N1亚型猪流感病毒、伪狂犬病毒、猪圆环病毒2型、猪细小病毒发生交叉反应。【结论】制备获得1株抗猪流感H3N2病毒HA蛋白的单克隆抗体,可用于猪流感病毒鉴别诊断方法的建立。

[Objective] To identify different swine influenza virus （SIV） and antibodies against SIV, monoclonal antibodies （Mabs） secreted by hybridoma ceil lines against SIV H3 subtype were prepared. [Method] Concentrated swine influenza A/swine/Henan/1/2OlO（H3N2） viruses were injected subcutane- ously or intraperitoneally into 6-week-old BALB/c mice with the rate of 0.2 mL per mouse according to the immunization protocol. For fusion, spleen cells and partner cells were washed, harvested, and mixed. Hybri- domas cells were screened using indirect ELISA method. Then, the monocolonal antibodies from hybrido- mas were tested by Western blot. [Result] One candidate hybridoma named 1C10 was identified after clone and purification by limiting dilution for three times. The titre of ascitic fluids of 1C10 was up to 1 ： 51 200. Monoclonal antibody 1C10 was IgG1 subtype and its light chain was kappa-type. Western blot showed that the MAb was specifically bounded to SIV H3N2 virus. 1C10 can be captured by hemagglutinin protein （HA） protein of SIV H3N2 in indirect ELISA and Western blot tests. There was no crossing reaction be- tween 1C10, SIV HIN1 subtype,PRRSV, PCV2, and PPV. [Conclusion] The obtained monoclonal antibody1C10 could be a useful reagent for diagnosing SIV infection and analyzing the epitopes of HA protein of SIV H3N2 virus.