猪繁殖与呼吸综合征病毒Nsp2的原核表达及单克隆抗体的制备

Expression of Nsp2 Gene of PRRSV and Development of Monoclonal Antibodies against Nsp2

以猪繁殖与呼吸综合征病毒(PRRSV)为模板,通过RT-PCR扩增编码非结构蛋白2(Nsp2)的基因,插入到pET32a表达载体中并在大肠埃希氏菌中进行表达。SDS-PAGE电泳结果显示融合表达的Nsp2分子量约为68 kDa,Western blotting分析该融合蛋白能与PRRSV阳性血清发生特异性反应。纯化融合表达产物Nsp2,按100μg/只的剂量与等量弗氏佐剂乳化,经腹腔免疫BALB/c小鼠3次后,取脾细胞与SP2/0骨髓瘤细胞进行融合,分别以Nsp2和His-Tag为抗原,通过间接EL ISA方法对融合细胞的上清液进行检测,筛选阳性克隆。经过3次亚克隆后,最终得到了6株能稳定分泌抗Nsp2抗体的阳性细胞克隆株。间接免疫荧光试验、Western blotting和间接ELISA试验鉴定这6株单克隆抗体均能够特异、单一地识别Nsp2,亚型鉴定结果显示6株单抗均属IgG2a型,其轻链均为κ链。所制备的这些单抗将为鉴定PRRSV Nsp2的B细胞抗原表位以及分析该蛋白的结构和功能奠定基础。

The gene fragment encoding Nsp2 of a North American-type PRRSV, was cloned into a prokaryotic expression vector pET-32a, and a fusion-expressed protein Nsp2 of 68KDa was obtained in Escherichia coll. The protein Nsp2 showed a strong reaction to the PRRSV- positive pig antisera in Western blotting analysis. The BALB/c mice were immunized hypodermically with 100 μg of Nsp2 fusion protein emulsified in Freund＇ s complete（ first injection） or incomplete adjuvant, which three injections at two week intervals. Splenocytes were fused with SP2/0 myeloma cells of the immunized mice after the third immunization. An indirect ELISA coated with Nsp2 and His-Tag was used to screen positive hybridomas for detection of specific antibody in culture supernatant of hybridoma. After three cycles of subclonlng, six hybridomas clones producing steadily McAbs against Nsp2 were obtained, which the subclass specificity were IgG2a with K light chain. All the six McAbs showed strong perinuclear fluorescence in PRRSV infected MARC-145 cells when used in immunofluorescence staining. Western blotting and indirect ELISA also indicated that the six McAbs were specific against the Nsp2. These McAbs could be used for identification of B-ceU epitopes on PRRSV Nap2, and may be helpful for further analysis of the structure and function of Nsp2.