【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...【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∶12800,1∶51200。【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。展开更多
The Tianshan orogenic belt is a major part of the southern Central Asian Orogenic Belt(CAOB),extending from west to east for over 2500 km through Uzbekistan,Tajikistan,Kyrgyzstan and Kazakhstan to Xinjiang in NW Chi...The Tianshan orogenic belt is a major part of the southern Central Asian Orogenic Belt(CAOB),extending from west to east for over 2500 km through Uzbekistan,Tajikistan,Kyrgyzstan and Kazakhstan to Xinjiang in NW China,and contains the record of multi-phase tectonothermal evolution.Till now.展开更多
基金supported by the Major Basic Research Project of the Ministry of Science and Technology of China(Grant No.2014CB448000)National Science Foundation of China(Grant Nos..41473053 and 41573045)a grant of Chinese Ministry of Land and Resources(Grant No.201211074–05)
文摘The Tianshan orogenic belt is a major part of the southern Central Asian Orogenic Belt(CAOB),extending from west to east for over 2500 km through Uzbekistan,Tajikistan,Kyrgyzstan and Kazakhstan to Xinjiang in NW China,and contains the record of multi-phase tectonothermal evolution.Till now.