摘要
研究旨在表达流行性出血病病毒(EHDV)VP7蛋白并制备其多克隆抗体,为EHDV检测方法的建立提供材料。试验通过大肠杆菌进行VP7重组蛋白的诱导表达,通过镍离子亲和层析与透析进行重组蛋白的纯化与复性并免疫家兔制备多克隆抗体,通过间接ELISA、Western blotting与间接免疫荧光试验(IFA)分析多克隆抗体的效价与反应原性。结果显示,在37℃与IPTG(0.1 mmol/L)的诱导下,VP7重组蛋白(分子质量46 ku)以包涵体形式高效表达,纯化与复性处理后的重组蛋白纯度达94.52%,可与不同血清型的EHDV阳性血清特异性结合。制备的兔抗VP7蛋白多克隆抗体效价达1∶24000;以制备的多克隆抗体为一抗,通过IFA与Western blotting检测到EHDV感染细胞中VP7蛋白的表达。本研究在大肠杆菌中实现了EHDV VP7蛋白的高效表达,制备的兔抗VP7蛋白多克隆抗体具有良好的反应原性与特异性,为EHDV诊断抗原的制备与血清学检测方法的建立提供了试验材料。
To provide basis for diagnostic methods of Epidemic hemorrhagic disease virus(EHDV),VP7 protein of EHDV was expressed and polyclonal antibody against the protein was prepared in present study.Recombinant VP7 protein expressed in E.coli was purified and renatured through Ni2+-NTA affinity chromatography and dialysis,respectively.Specific polyclonal antibodies against the expressed VP7 protein were obtained by immunized rabbits.The titer and reactogenicity of the prepared antibodies were evaluated through indirect ELISA,Western blotting and indirect immunofluorescence assay(IFA),respectively.The results showed that VP7 recombinant protein with molecular weight at approximately 46 ku was efficiently expressed in E.coli as inclusion body when induced at 37℃with the concentration of IPTG at 0.1 mmol/L.After purification and renaturation,the purity of recombinant VP7 protein was 94.52%,which could be recognized by sera of different EHDV serotypes.The titer of the prepared rabbit polyclonal antibody against EHDV VP7 was 1∶24000 and the VP7 protein expressed in the EHDV infected cells was detected by Western blotting and IFA using the prepared antibody.In summary,VP7 protein of EHDV was successfully expressed in E.coli and rabbit polyclonal antibody against VP7 protein with robust specificity and reactivity was prepared,which provided basic materials for the development of EHDV diagnostic antigen and the establishment of serological detection method.
作者
李占鸿
宋子昂
肖雷
朱建波
杨振兴
李卓然
廖德芳
李华春
杨恒
LI Zhanhong;SONG Ziang;XIAO Lei;ZHU Jianbo;YANG Zhenxing;LI Zhuoran;LIAO Defang;LI Huachun;YANG Heng(Yunnan Tropical and Subtropical Animal Viral Disease Laboratory,Yunnan Animal Science and Veterinary Institute,Kunming 650224,China;College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China)
出处
《中国畜牧兽医》
CAS
北大核心
2021年第4期1405-1413,共9页
China Animal Husbandry & Veterinary Medicine
基金
国家重点研发计划(2016YFD0500908)
公益性行业(农业)科研专项(201303035)
云南省中青年学术和技术带头人后备人才培养项目(2017HB055)。
