美洲型猪繁殖与呼吸综合征病毒GP4蛋白中和活性单克隆抗体制备及其抗原表位鉴定

Preparation of a monoclonal antibody with neutralizing activity against North American PRRSV GP4 and identification of its epitope

为制备具有中和活性的抗猪繁殖与呼吸综合征病毒(PRRSV)的单克隆抗体(MAb)并鉴定其抗原表位,本研究利用PRRSVSD53株的超离病毒作为免疫原,按常规方法免疫BALB/c小鼠并进行细胞融合。采用PRRSVSD53株和真核表达的PRRSV各结构蛋白作为检测抗原,应用间接免疫荧光试验(IFA)筛选到一株稳定分泌抗SD53株GP4蛋白的杂交瘤细胞株(LM26)。抗体亚类鉴定其为IgG2a,轻链为κ链。原核表达一系列截短的GP4蛋白鉴定LM26的抗原表位结果显示,LM26识别的抗原表位序列为57VVLQDI^62,此抗原表位在美洲型PRRSV中相对保守。病毒中和试验结果显示,MAb LM26仅针对PRRSV SD53株具有中和活性,中和效价最高为1∶50,对其它美洲株PRRSV均不具有中和活性。该MAb的制备为进一步研究PRRSVGP4蛋白的结构和功能奠定了基础。

In order to prepare a monoclonal antibody (MAb) against porcine reproductive and respiratory syndrome virus (PRRSV) with neutralizing activity, BALB/c mice were immunized with ultracetrifugate-purified PRRSV SD53 strain, and cell fusion was performed following a routine method. The recombinant structural proteins of PRRSV were used as the detection antigen to screen the positive hybridoma cells by an indirect immunofluorescence assay. One hybridoma cell line (LM26) that can stably secrete MAb against GP4 protein of SD53 strain was obtained. The antibody subclass was identified as IgG2a, and light chain was κ chain. A series of truncated GP4 proteins were expressed by prokaryotic expression system, and the epitope recognized by the MAb LM26 was identified as 57VVLQDI^62 of GP4. This epitope was relatively conservative among different North American PRRSV strains. Virus neutralization test illustrated that MAb LM26 had neutralizing activity only against PRRSV SD53 strain at the titer of 1:50, but it did not have neutralizing activity against other American strains. The MAb prepared in this study provides a basis for further study on structure and function of PRRSV GP4.