| 王雅慧，王英英，王 庆，李 波，李莹莹，尹纪元，杨 广，曾伟伟.罗非鱼湖病毒 S10 基因编码蛋白的表达及单克隆抗体的制备[J].广东农业科学,2021,48(11):112-118 |
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罗非鱼湖病毒 S10 基因编码蛋白的表达及单克隆抗体的制备 |
| Encoding Protein Expression of S10 Gene of Tilapia Lake Virus and Preparation of Monoclonal Antibody |
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| DOI：10.16768/j.issn.1004-874X.2021.11.014 |
| 中文关键词: 罗非鱼 罗非鱼湖病毒（TiLV） S10 编码蛋白 重组蛋白 单克隆抗体 免疫学方法 |
| 英文关键词: tilapia Tilapia Lake Virus（TiLV） S10 encoded protein recombinant protein monoclonal antibody immunological method |
| 基金项目:国家重点研发计划项目（2019YFD0900101）；广东省基础与应用基础研究基金（2020A1515010327）；广东省现代农业产业技术体系创新团队建设专项（2021KJ119）；国家大宗淡水鱼产业技术体系（CARS-45） |
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| 中文摘要: |
| 【目的】制备抗罗非鱼湖病毒（Tilapia Lake Virus, TiLV）S10节段基因编码蛋白的单克隆抗体（Monoclonal antibody，MAb）。【方法】构建含有TiLV S10基因节段的重组表达质粒pET32a -S10，并将其转化至BL21（DE3）感受态细胞，经IPTG诱导表达和镍柱纯化获得高纯度重组S10蛋白；用纯化的重组蛋白免疫6~8周龄雌性BALB/c小鼠多次后，取其脾细胞与SP2/0细胞进行融合获得杂交瘤细胞，采用有限稀释法和ELISA方法，筛选获得2株能稳定分泌抗S10蛋白MAb的杂交瘤细胞株，分别命名为2C3和2E3，并对其特异性、亚型和效价进行分析。【结果】Western blot结果显示，2C3和2E3均能识别S10重组蛋白及TiLV，间接免疫荧光试验（IFA）结果表明，2C3和2E3只与TiLV感染的TiB细胞呈阳性反应。亚型检测结果显示，2C3抗体为IgG1/к型，2E3抗体为IgG2a/к型。抗体效价测定结果表明，两种MAb的效价分别为 1∶12 800，1∶51 200。【结论】抗TiLV-S10蛋白MAb特异性强、效价高，可为后续TiLV疫苗的研发、免疫学方法的建立及S10蛋白功能的研究提供重要材料和支撑。 |
| 英文摘要: |
| 【Objective】The study was carried out to prepare the monoclonal antibodies（MAb）of S10-segmentcoded protein against Tilapia Lake Virus（TiLV）.【Method】A recombinant expression plasmid pET32a -S10 that could efficiently express the TiLV S10 gene segment was constructed and it was converted into BL21（DE3）competent cells, which were induced by IPTG expression and purified by nickel column to obtain recombinant S10 protein with high purity. Female BALB/c mice aged 6-8 weeks were immunized with purified S10 protein to fuse their spleen cells with SP2/0 cells to obtain hybridoma cells. Two hybridoma cell lines that could steadily secrete anti-S10 protein MAb were screened by finite dilution and ELISA method. The hybridoma cells were named 2C3 and 2E3 respectively, and their specificity, subtype and titer were analyzed.【Result】Western blot results show that both 2C3 and 2E3 can recognize S10 recombinant protein and TiLV. Indirect immunofluorescence assay（IFA）results show that 2C3 and 2E3 only react positively with TiB cells infected with TiLV. Subtype detection results show that 2C3 antibodies are IgG1/к and 2E3 antibodies are IgG2a/к. Indirect ELISA shows that the titers of the two MAbs are 1:12 800 and 1:51 200, respectively.【Conclusion】The anti-TiLV-S10 protein MAb is specific and highly effective, which can provide materials and support for the development of TiLV vaccine, the establishment of immunological methods and the study of S10 protein function. |
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