裂谷热(RVF)是由裂谷热病毒(RVFV)引起的一种人兽共患传染病,主要由蚊媒传播.该病主要感染反刍动物,可引起流产和新生胎儿死亡,人类对该病也易感,严重者可导致死亡.对于无RVF的国家,建立相应的病原学与血清学检测方法对于防止该病传入至关重要.为了建立基于病毒N蛋白的诊断技术,本研究构建了裂谷热病毒N基因原核表达质粒pET-28a-N,转化BL21(DE3),IPTG诱导表达,通过SDS-PAGE和Western blot检测蛋白的表达,重组蛋白分子量约为29 kDa,主要以上清液形式存在.大量表达并纯化重组蛋白,用纯化后的重组蛋白免疫BALB/c小鼠,制备单克隆抗体,并对其进行鉴定.采用间接ELISA和ID Vet公司阻断ELISA试剂盒检测免疫小鼠抗体效价及特异性,并用Western blot检测多克隆抗体反应性.结果表明,免疫后的抗体效价迅速升高,最终可达51 200以上;阻断ELISA和Western blot检测结果表明,制备的多克隆抗体具有良好的反应性与特异性.通过3次亚克隆最终获得2株分泌单克隆抗体的杂交瘤细胞株,分泌的单克隆抗体均可与重组蛋白反应,重链均为IgG2a型,轻链为κ型.本研究为RVF诊断方法的建立奠定了基础.

为了对疑似污染牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的牛肾细胞系(MDBK)进行检测并分离鉴定病毒,采用RT-PCR、间接免疫荧光、电镜观察、病毒序列的分子进化分析等方法对细胞中污染的BVDV进行鉴定.结果显示:从2株不同来源的污染细胞中检测到2株BVDV(C201602和C201704),RT-PCR均可扩增得到288 bp的5'-UTR片段,进化分析表明其均为BVDV-2a型;2株病毒接种MDBK细胞传代,均无细胞病变产生,测得第3代病毒效价分别为105和104.5 TCID5o/mL,电镜观察病毒为50 ～ 60 nm的圆形有囊膜颗粒.研究表明,BVDV-2具有潜在的流行风险,为试验材料中BVDV-2污染的风险控制提出了警示.

With the development of the cattle breeding industry,bovine respiratory disease complex (BRDC) is becoming more and more prevalent.The disease is caused by co-infection of multiple viruses and bacteria.Under the change of immune status and/or environmental conditions,severe symptoms could be developed,resulting in high morbidity and even mortality.In this study,the causative pathogens of a BRDC case of one beef cattle farm in Jiangsu Province were identified by means of field survey,clinical observation,pathological examination and pathogen detection.Co-infection of bovine viral diarrhea virus (BVDV),bovine parainfluenza virus type 3 (BPIV3) and Escherichia coli was finally identified,and measures and guidelines for prevention and control of the disease were proposed.