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| 刀豆蛋白A纯化猪流行性腹泻病毒的研究 |
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李兰1,张浩明1,杨利1,程海卫1,侯立婷1,张元鹏1,侯继波1,陈瑾1,郑其升1,2,3*
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| (1.江苏省农业科学院 动物免疫工程研究所, 南京 210014;2.江苏省食品质量安全重点实验室国家重点实验室培育基地, 南京 210014;3.兽用生物制品(泰州)国泰技术创新中心, 泰州 225300) |
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| 摘要: |
| 为开发一种新型高效、低成本的猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV)抗原纯化方法，本研究尝试了一种基于凝集素(刀豆蛋白A，Con A)的PEDV浓缩纯化方法。通过Con A与PEDV病毒粒子的特异性结合，形成Con A-PEDV复合物，然后通过低速离心和竞争性解离即可实现病毒纯化。结果表明:1)当Con A的使用浓度为80 μg/mL，室温作用2 h，4 000 r/min离心15 min时，纯化效果较佳；2)利用Tris-HCl缓冲液(pH 7，0.01 mol/L，内含200 mmol/L甲基-α-D-吡喃甘露糖苷和50 mmol/L NaCl)进行解离时，较PBS缓冲液有更高的解离效率；3)该方法病毒回收率大约为80%，总蛋白去除率可达90%。综上，本研究利用Con A纯化PEDV是可行的，为高质量PEDV灭活疫苗的研发奠定基础。 |
| 关键词: 刀豆蛋白A 猪流行性腹泻病毒 纯化 |
| DOI：10.11841/j.issn.1007-4333.2023.11.11 |
| 投稿时间：2022-12-27 |
| 基金项目:国家自然科学基金项目(32102690)；江苏省农业科技自主创新资金(CX(21)3135) |
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| Research on the purification of porcine epidemic diarrhea virus by Concanavalin A |
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LI Lan1,ZHANG Haoming1,YANG Li1,CHENG Haiwei1,HOU Liting1,ZHANG Yuanpeng1,HOU Jibo1,CHEN Jin1,ZHENG Qisheng1,2,3*
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| (1.National Research Center of Engineering and Technology for Veterinary Biologicals, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;2.Cultivation Base of State Key Laboratory for Food Quality and Safety of Jiangsu Province, Nanjing 210014, China;3.GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China) |
| Abstract: |
| In order to develop a new efficient and low-cost method for antigen purification of porcine epidemic diarrhea virus(PEDV), a purification method based on the lectin(Concanavalin A, Con A)was explored. By specifically binding Con A with PEDV virus particles, a Con A-PEDV complex was formed, and the virus was purified through low-speed centrifugation and competitive dissociation. The results showed that: 1)The suitable concentration of Con A was 80 μg/mL, the action time between Con A and PEDV was for 2 hours, the centrifugal condition was 4 000 r/min for 15 min; 2)The solution contained methyl-α-D-mannopyranoside and NaCl at the concentration of 200 mmol/L and 50 mmol/L respectively had higher dissociation efficiency compared with PBS buffer; 3)The recovery rate of the purification method was approximately 80%, and the total protein removal rate could reach 90%. In conclusion, it is feasible to purify PEDV by Con A, which can lay a foundation for the development of high-quality inactivated vaccine for PEDV. |
| Key words: Concanavalin A porcine epidemic diarrhea virus purification |
