猪瘟E^rns蛋白在杆状病毒表达系统中的表达及其抗体间接ELISA方法的建立  

Expression of the classical swine fever virus E^rns in baculovirus expression system and the establishment of an indirect ELISA

作　　者：任杰 葛猛[1] 李润成[1] 郑金[1] 凌同 黎满香[1] 李岱霞 张磊[1] 余兴龙[1] REN Jie;GE Meng;LI Run-cheng;ZHENG Jin;LING Tong;LI Man-xiang;LI Dai-xia;ZHANG Lei;YU Xing-long(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Engineering Technology Center,Hunan Sinoland Biological Pharmaceutical Co.,Ltd.,Changsha 410100,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]湖南中岸生物药业有限公司工程技术中心,湖南长沙410100

出　　处：《中国兽医学报》2020年第3期450-456,共7页Chinese Journal of Veterinary Science

基　　金：湖南省教育厅一般项目(18C0165);国家“十三五”重点研发计划资助项目(2017YFD0500102)。

摘　　要：将人工合成的CSFV E^rns基因插入到pFastBac1获得重组转移载体pFastBac1-E^rns,在DH10Bac大肠杆菌中转座后提取杆粒并转染Sf9细胞,用获得的重组杆状病毒感染High Five细胞表达E^rns蛋白并通过亲和层析进行纯化。以纯化后E^rns蛋白作为诊断抗原,通过摸索抗原包被量和抗体血清稀释倍数等条件,建立检测CSFV E^rns抗体的间接ELISA方法(E^rns-iELISA)。结果显示,E^rns蛋白得到有效表达,相对分子质量约为35000;确定的ELISA抗原包被量为120ng/孔、血清稀释100倍,酶标二抗稀释6000倍,封闭条件为37℃30min,TMB底物作用15min,临界值为0.329。用E^rns-iELISA方法检测92份阴性血清,其特异性为88.04%,与猪圆环病毒2型、猪繁殖与呼吸系统综合征病毒和猪流行性腹泻病毒阳性血清均无交叉反应;对171份阳性血清的敏感性为92.98%;批内和批间重复的平均变异系数为7.4%和10.74%。结果表明,本试验所建立的E^rns-iELISA方法重复性好,具有较高的检测特异性,且对CSFV疫苗毒和国内流行毒株都具有较高的检测敏感性,具有潜在的开发和应用价值。Recombinant donor vector pFastBac-E^rns was obtained by inserting synthesized CSFV E^rns gene into pFastBac1,after transposition in DH10Bac E.coli,rBacmids were extracted and transfected into Sf9cells.E^rns protein was expressed in High Five cells infected with the obtained recombinant baculovirus and purified by affinity chromatography.An indirect ELISA method was established using purified E^rns protein by exploring the concentration of coated antigen and dilution of serum samples.E^rns protein was expressed with a molecular weight of about 35000.The optimal volume of coated antigen and serum dilution was 25ng each well and 100times;the second antibody dilution was 6000times;closure condition was 37℃1h;TMB incubation condition was 15min and critical value was 0.329.Specificity of E^rns-iELISA was 88.04%showed in detecting of 92negative serum samples and there was no cross-reactivity to porcine circovirus type 2,porcine reproductive and respiratory syndrome virus and porcine epidemic diarrea virus positive serum.The sensitivity of E^rns-iELISA was 92.98%showed in detecting of 171positive serums.The average coefficient of variation was 7.4%and 10.74%in intra-assay and inter-assay.The E^rns-iELISA method established in this study has potential development and application value owing to its good repeatability,high specificity,and high sensitivity to CSFV vaccine virus and domestic epidemic strains.

关 键 词：猪瘟病毒 E^rns蛋白 杆状病毒表达系统 抗体 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]