摘要
非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种危害性极强的病毒性疾病,该病的发生和流行对全球养猪业、猪肉食品供应及经济发展造成了巨大影响。ASFV基因组较大、编码蛋白众多,但目前很多病毒蛋白的功能尚不清楚。由于缺乏有效的治疗措施和安全的疫苗,所以ASF的防控及疫区的净化仍然面临巨大挑战,而早期、快速、可靠的实验室诊断仍然是该病防控的重要手段。本研究以ASFV基因Ⅱ型毒株的基因组为模板,扩增A104R、B602L、CP204L (P30)、B646L(P72)和K205R基因,并将其克隆至表达载体pET-30a,构建原核表达质粒,经IPTG诱导后表达5种病毒蛋白,其中p A104R、pK205R为可溶性表达,P30、P72、pB602L为包涵体表达。经Western-blot分析鉴定蛋白的抗原性,五种原核表达的蛋白都能够和ASF阳性血清发生特异性反应,通过比较,筛选出pA104R作为最佳诊断抗原,并初步建立了ASFV抗体检测间接ELISA方法。该方法能够敏感、特异的检测ASFV阳性血清,而与猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)、口蹄疫病毒(FMDV)阳性血清均无交叉反应。利用建立的间接ELISA方法与商品化间接ELISA抗体检测试剂盒平行检测田间样本,结果表明,本研究建立的方法与商品化试剂盒之间的符合率达到94.7%(142/150)。上述结果表明,本研究建立的间接ELISA方法能够用于ASF血清抗体的检测。
African swine fever(ASF) is a serious viral disease caused by African swine fever virus(ASFV),it posed a great threat to the global pig industry,food safety and economy.Due to the lack of safe and effective treatments and vaccines,the prevention,control and the eradication of epidemic areas of ASF are still facing huge challenges.ASFV has a large genome and encodes many proteins and their functions are currently unclear.In this study,the A104 R,B602 L,CP204 L,B646 L and K205 R gene were amplified based on genotype II of ASFV,and recombinant plasmids pE T-A104 R,-K205 R,-CP204 L,-B602 L,and-B646 L were constructed through ligation and transformation.Recombinant proteins were induced by IPTG and purified by nickel column.The expression of proteins was identified by Western-blotting.The results showed that the five recombinant proteins had been expressed successfully and had specific reaction with ASFV positive sera.The A104 R showed the best antigen reactivity and was selected for the development of an indirect ELISA serological diagnostic method.No cross-reaction was detected with positive sera of porcine reproductive and respiratory syndrome virus,classic swine fever virus,porcine circovirus type 2,porcine pseudorabies virus,and foot-and-mouth disease virus by this method.A total of150 field sera samples were collected and detected in parallel by using the indirect ELISA and the commercial indirect ELISA Kit,the coincidence rate between them was 94.7%(142/150).These results showed that this indirect ELISA method has high specificity and sensitivity,and can be used for the serological diagnosis of ASF.
作者
王兆贵
赵亚茹
杨吉飞
牛庆丽
刘银光
敬梦瑶
杨赛霞
吕欣倩
刘猛
姚永海
关贵全
刘志杰
殷宏
WANG Zhao-gui;ZHAO Ya-ru;YANG Ji-fei;NIU Qing-li;LIU Yin-guang;JING Meng-yao;YANG Sai-xia;LU Xin-qian;LIU Meng;YAO Yong-hai;GUAN Gui-quan;LIU Zhi-jie;YIN Hong(African Swine Fever Regional Laboratory of China(Lanzhou)/State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Baishuijiang Nature Reserve Administration;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis,Yangzhou University,Yangzhou 225009,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2021年第4期403-411,共9页
Chinese Veterinary Science
基金
国家自然科学基金项目(31941012)
中国农业科学院基本科研业务费专项(Y2019YJ07-02)
中国农业科学院农业科技创新工程项目(CAAS-ASTIP-2016-LVRI)
甘肃省重点研发计划项目(18YF1WA131)
甘肃省重大专项(20ZD7NA006)。
关键词
非洲猪瘟
非洲猪瘟病毒
蛋白纯化
间接ELISA
African swine fever
African swine fever virus
protein purification
indirect ELISA
