【作者】 李文超；

【导师】 章金刚； 吴斌； 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 蓝舌病（Bluetongue disease，BT）是由蓝舌病病毒（Bluetongue virus，BTV）引起，由库蠓传播的反刍动物病毒性传染病。世界动物卫生组织（OIE）将其列为A类动物疫病。该病主要集中在各热带、亚热带和温带国家，给畜牧业造成巨大的损失。在蓝舌病病毒编码的各种蛋白质中，VP7蛋白为群特异性抗原。VP7蛋白是蓝舌病病毒主要的核心抗原，携带有群特异性抗原决定簇，能刺激被感染机体产生强的群特异性免疫反应。因此，VP7基因已成为BTV研究的热点。本研究在克隆并鉴定蓝舌病病毒VP7基因基础上，进行VP7蛋白的原核表达，并制备相应单克隆抗体，为诊断试剂研制奠定了基础。1．蓝舌病病毒VP7蛋白的原核表达及纯化产物反应原性鉴定根据已发表BTV VP7基因序列，设计并合成一对引物。利用RT-PCR方法扩增BTV-5型毒株编码群特异性抗原的VP7基因片段。将扩增片段克隆至pGEM-T Easy载体中，构建VP7基因重组载体。经核苷酸序列测定，克隆的基因片段长1050bp，为S7基因开放性读码框的全长序列，编码349个氨基酸。将VP7基因片段连接至pET-DsbA、pET-GST和pET-His表达载体中，经抗性筛选、PCR扩增、限制性内切酶分析，筛选获得BTV VP7基因片段正向插入、有正确读码框的阳性克隆。阳性克隆质粒在E.coli BL21（DE3）pLysS感受态细胞中经IPTG诱导表达，筛选到高表达重组载体pET-DsbA／VP7。镍琼脂糖凝胶亲和层析分离重组载体pET-DsbA／VP7表达的带His标签的重组VP7蛋白。SDS-PAGE、Western blotting和ELISA实验表明，表达蛋白为融合蛋白，分子量约为63.2kDa，具有良好的群特异反应原性。2．蓝舌病病毒VP7蛋白的单克隆抗体制备及鉴定本研究通过BHK-21接种，增殖蓝舌病病毒，将收获的病毒液用甲醛灭活。用纯化病毒免疫Balb／c小鼠，取免疫鼠脾细胞与SP2／0骨髓瘤细胞融合。用ELISA试验筛选阳性株，采用有限稀释法对其进行克隆，经过融合共获得2株能稳定分泌抗BTV VP7蛋白单克隆抗体的杂交瘤细胞株，分别命名为3E2和1C11。细胞培养上清ELISA效价分别为2⁹和2¹⁰，腹水ELISA效价可达4×10⁶和16×10⁶。所有单抗特异性良好，与鹿流行性出血热病毒、背景源蛋白无交叉反应。腹水中纯化的单抗3E2与不同BTV血清型的绵羊抗血清进行竞争ELISA反应，证实有良好的竞争抑制性。更多还原

【Abstract】 Bluetongue disease （BT） is an arthropod-borne viral disease affecting ruminants,caused by Bluetongue virus （BTV）. At present, bluetongue disease is listed in A infectiousdiseases by World Organization for Animal Health （OIE）. The disease broke out primarilyin tropical and temperate regions of the world, and resulted in a serious economic loss.VP7 is the major core antigen of bluetongue virus, which can induce the production ofgroup-specific antibodies against the virus. And there are group-specific antigenic sitesmapped on the VP7. In this study group-specific antigen VP7 of BTV was expressed inE.coli by a recombinant vector, and highly expressed recombinant VP7 for serologicaltests of BTV as a diagnostic reagent antigen. Two McAbs against group-specific antigenVP7 of BTV were prepared. This study gives us a clue to research the diagnosis reagent.1. Expression and antigentic characterization of the major core protein VP7 ofBluetongue virusAccording to published VP7 gene sequence of BTV, primers were designed andsynthesized. The complete cDNA clone of VP7 gene in the cloning procedure wasamplified by RT-PCR. The group-specific antigen VP7 gene of BTV was cloned intopGEM-T Easy vector and sequenced. The length of VP7 gene was 1050 bp, whichencoded 349 amino acids. The recombinant vector containing BTV VP7 cDNA wasconstructed. Then the fragment of VP7 gene was subcloned into prokaryotic expressionvector pET-DsbA、pET-GST and pET-His by PCR and restriction endonucleases. Therecombinant expression vectors were transformed into E.coli BL21 （DE3） pLysS andinduced by IPTG. The higher expression vector pET-DsbA/VP7 was screened. TheHis-tagged recombinant protein was purified using a His-tag affinity chromatographycolumn on Ni²⁺-nitrilotriacetate （NTA） resin. The results of SDS-polyarcylamide gelelectrophoresis （SDS-PAGE） and Western blotting revealed that the recombinant fusionprotein was expressed with a molecular mass of approximately 63.2 kDa. The indirectenzyme-linked immunosorbent assay （ELISA） indicated that the expressed BTV VP7 washighly immunogenic.2. Preparation of Monoclonal Antibodies against serogroup-specific antigen VP7 ofBluetongue virusBTV-5 was cultured in BHK-21 cell. The obtained virus was inactivated with 0.1% formaldehyde. Balb/c mice were immunized with the purified BTV antigens. Splenocytesfrom the immunized mice were fused with SP2/0 myeloma cells, and positive hybridomaclones were screened by ELISA and limited dilution method was performed to subclonethe positive clones. After three cycles of subcloning, two McAbs against the VP7 ofBTV-5 were obtained, designated as 3E2 and 1Cll. The ELISA titer of culturesupernatant were 2⁹ and 2¹⁰, and ascites were 4×10⁶ and 16×10⁶, respectively. TheseMcAbs had good specificity to BTV-5. No cross-reactions were found when epizootichemorrhagic disease virus （EHDV）, BHK-21 cell, and E.coli cell were tested. McAb 3E2purified from wild waterfowl had high activity of binding the recombinant fusion proteinVP7, detected by competitive ELISA with different serotypes of sheep anti-BTVantibodies.更多还原