【摘要】 非结构蛋白nsp4是马动脉炎病毒（EAV）复制过程中的关键蛋白之一，参与病毒复制及调控宿主的天然免疫反应。为制备EAV nsp4蛋白的单克隆抗体（MAb），本研究分别构建重组质粒pET32a-nsp4-his和pGEX-6p-1-nsp4-GST，经测序鉴定正确后采用原核系统表达两种重组nsp4蛋白（nsp4-his、nsp4-GST），采用his和GST亲和层析柱分别纯化这两种蛋白后经SDS-PAGE检测。结果显示，分别于42 ku与50 ku处出现nsp4-his和nsp4-GST蛋白条带，均为可溶性表达，且二者的纯化效果均较好。采用纯化的nsp4-his蛋白免疫BALB/c小鼠，三免后以nsp4-GST作为包被抗原，采用间接ELISA法测定小鼠血清的抗体效价。选择抗体效价最高的小鼠冲击免疫，3 d后取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合，通过上述间接ELISA筛选稳定分泌nsp4蛋白MAb的杂交瘤细胞。结果显示，三免后小鼠血清抗体效价最高达1:1.28×10⁵，表明该蛋白的免疫原性较强。经间接ELISA筛选，结果显示，获得6株稳定分泌nsp4蛋白MAb的杂交瘤细胞株。取间接ELISA检测OD_450nm值最高的杂交瘤细胞制备腹水并采用亲和层析方法纯化后，采用Western blot与间接免疫荧光试验（IFA）检测MAb的反应性。结果显示，不同稀释度的MAb与细胞中过表达的及EAV感染细胞中表达的nsp4蛋白反应后均在25 ku处出现特异性条带，且感染EAV的RK13细胞中出现绿色荧光，未感染EAV的阴性对照细胞无绿色荧光。将EAV以MOI 1的剂量感染RK13细胞，于感染后不同时间采用激光共聚焦试验检测nsp4蛋白与线粒体的定位。结果显示，在EAV感染后不同时间，代表nsp4蛋白的红色荧光与代表线粒体外膜转位酶复合体亚基20蛋白（TOM20）的绿色荧光均出现于胞质内，二者在细胞质中融合后形成黄色的荧光，阴性对照无荧光。上述结果表明，本研究获得具有良好免疫原性的nsp4蛋白，制备了具有良好反应性的nsp4 MAb，并利用该MAb首次检测到EAV nsp4蛋白与线粒体在胞质内存在共定位，本研究为深入探究EAV nsp4蛋白的功能及EAV的生物学特性提供了生物材料。更多还原

【Abstract】 Non-structural protein nsp4 is one of the key proteins in the replication process of equine arteritis virus（EAV）,involved in viral replication and regulation of the host’s innate immune response. To prepare monoclonal antibodies（MAbs） against EAV nsp4 protein, recombinant plasmids p ET32a-nsp4-his and pGEX-6p-1-nsp4-GST were constructed in this study. After confirmation by sequencing, the two recombinant nsp4 proteins（rnsp4） were expressed using a prokaryotic system. The nsp4-His and nsp4-GST proteins were purified using His and GST affinity chromatography columns, respectively, and identified by SDS-PAGE.Results showed protein bands for nsp4-His and nsp4-GST at approximately 42 ku and 50 ku, respectively, both of which were soluble and exhibited good purification efficiency. BALB/c mice were immunized with the purified nsp4-His protein. After the tertiary immunization, the antibody titer in mouse serum was determined using an indirect ELISA method with nsp4-GST as the coating antigen.The mouse with the highest antibody titer was selected for booster immunization. Three days later, splenocytes from the immunized mouse were fused with SP2/0 myeloma cells. Hybridoma cells stably secreting MAbs against the nsp4 protein were screened using the aforementioned indirect ELISA. The results showed that the highest serum antibody titer after the tertiary immunization reached1:1.28×10⁵, indicating strong immunogenicity of the protein. Indirect ELISA screening results showed that six hybridoma cell lines stably secreting MAbs against the nsp4 protein were obtained. The hybridoma cell line with the highest OD_{450 nm}value in indirect ELISA was selected for ascites production, and the MAbs were purified using affinity chromatography. The reactivity of the MAbs was evaluated by Western blot and indirect immunofluorescence assay（IFA）. The results showed that MAbs at different dilutions reacted with both overexpressed nsp4 protein in cells and nsp4 protein expressed in EAV-infected cells, producing specific bands at25 ku. Green fluorescence was observed in EAV-infected RK13 cells, while no green fluorescence was detected in uninfected negative control cells. RK13 cells were infected with EAV at an MOI of 1, and the localization of nsp4 protein and mitochondria was detected by confocal laser scanning microscopy at different time points post-infection. The results showed that at different times after EAV infection, the red fluorescence representing the nsp4 protein and the green fluorescence representing the mitochondrial outer membrane protein TOM20 were both located in the cytoplasm, producing yellow fluorescence. Uninfected cells had no fluorescence.The above results indicate that this study successfully obtained nsp4 protein with good immunogenicity and prepared nsp4 MAbs with good reactivity. Using these MAbs, co-localization of EAV nsp4 protein and mitochondria in the cytoplasm was detected for the first time. This study provides essential biological materials for further investigation of the function of EAV nsp4 protein and the biological characteristics of EAV.更多还原