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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (11): 35-42    DOI: 10.13523/j.cb.2006045
技术与方法     
猪圆环病毒2型病毒样颗粒的高效组装技术研究*
王彦伟,李鹏昊,梁严予,关洋,逄文强(),田克恭()
国家兽用药品工程技术研究中心 洛阳 471000
Efficient Assembly of Virus-like Particles of Porcine Circovirus Type 2
WANG Yan-wei,LI Peng-hao,LIANG Yan-yu,GUAN Yang,PANG Wen-qiang(),TIAN Ke-gong()
National Research Center for Veterinary Medicine, Luoyang 471000, China
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摘要：

目的:探索猪圆环病毒2型(PCV2)病毒样颗粒(VLPs)的高效组装技术,提高VLPs的稳定性。方法:利用大肠杆菌表达PCV2 Cap蛋白自组装为VLPs,分析不同离子强度下VLPs的稳定性。利用切向流技术添加尿素,降低pH,可使VLPs解组装,利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。结果:PCV2 Cap蛋白自组装VLPs在150mmol/L NaCl下稳定性较差,而在500mmol/L NaCl下可提高VLPs的稳定性,但仍较易发生聚集,核酸含量均较高。在150mmol/L NaCl、300mmol/L尿素和pH 5.5的缓冲体系条件下,能够使VLPs解组装。经25%~50%饱和硫酸铵(V/V)分级沉淀粗纯,阴离子交换层析500mmol/L NaCl下洗脱获得精纯Cap蛋白,蛋白质纯度≥95%,并能够有效去除核酸。通过切向流技术去除体系中的尿素,并将NaCl浓度提高至1mol/L、pH提高至8.0,改变蛋白质表面静电荷分布,实现VLPs的高效、均一再组装,组装效率≥99%,回收率为65.85%,并明显提高VLPs的稳定性,能够稳定保存6个月以上。结论:利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。
关键词： 猪圆环病毒2型;  病毒样颗粒;  切向流技术;  高效组装;  稳定性    
Abstract:

Objective: To explore the efficient assembly technology of virus-like particles (VLPs) of porcine circovirus type 2 (PCV2) and improve the stability of VLPs. Methods: PCV2 Cap protein was expressed in E. coli and self-assembled into VLPs. The stability of VLPs under different ionic strength was analyzed. Disassembly of VLPs was achieved by addition of urea and decreasing pH with tangential flow filtration. Cap protein was obtained by ammonium sulfate precipitation and anion exchange chromatography. Efficient reassembly of VLPs was achieved by removing urea,increasing ionic strength and pH. Results: The stability of self-assembled PCV2 VLPs was poor under 150mmol/L NaCl, and was improved under 500mmol/L NaCl, but it was still easy to aggregate. The nucleic acid content was high. Under the condition of 150mmol/L NaCl, 300mmol/L urea and pH 5.5, VLPs was disassembled. The crude protein was precipitated by 25%-50% saturated ammonium sulfate (V/V) and eluted by anion exchange chromatography under 500mmol/L NaCl to obtain the purified Cap protein with over 95% purity and 65.85% recovery, which the nucleic acid was effectively removed. Urea was removed, the concentration of NaCl was increased to 1mol/L, and the pH was increased to 8.0 with tangential flow technology. The static charge distribution on the protein surface was changed, and efficiently and uniformly reassembly of VLPs was achieved with over 99% assembly efficiency. The stability of VLPs was significantly improved, and was stored stably for more than six months. Conclusion: PCV2 cap protein was obtained by ammonium sulfate fractional precipitation and anion exchange chromatography. Then, the urea was removed, the ionic strength and pH were improved to realize the efficient reassembly of VLPs.
Key words: Porcine circovirus type 2    Virus-like particles    Tangential flow filtration    Efficient assembly    Stabilization
收稿日期: 2020-06-04 出版日期: 2020-12-11
ZTFLH:  Q819  
基金资助: * 郑洛新自创区创新引领型产业集群专项(201200211200)
通讯作者: 逄文强,田克恭     E-mail: pangwq123@hotmail.com;vetvac@126.com
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引用本文:

王彦伟,李鹏昊,梁严予,关洋,逄文强,田克恭. 猪圆环病毒2型病毒样颗粒的高效组装技术研究*[J]. 中国生物工程杂志, 2020, 40(11): 35-42.

WANG Yan-wei,LI Peng-hao,LIANG Yan-yu,GUAN Yang,PANG Wen-qiang,TIAN Ke-gong. Efficient Assembly of Virus-like Particles of Porcine Circovirus Type 2. China Biotechnology, 2020, 40(11): 35-42.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2006045    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I11/35

图1  PCV2 rCap蛋白的纯化和自组装VLPs
图2  自组装PCV2 VLPs的稳定性
图3  PCV2 VLPs的解组装
图4  解组装PCV2 rCap蛋白的纯化结果
样品类型 体积(ml) 总蛋白质浓度
(mg/ml)		 总蛋白质
(mg)		 rCap蛋白浓度
(mg/ml)		 总rCap蛋白
(mg)		 回收率(%)
裂解液上清 80 6.12 489.6 0.41 32.8 100
硫酸铵粗纯 80 1.68 134.4 0.34 27.2 82.93
自组装VLPs 20 1.23 24.6 1.14 22.8 69.51
解组装rCap蛋白 20 1.20 24 1.08 21.6 65.85
表1  纯化阶段PCV2 rCap蛋白的回收率
图5  PCV2 VLPs的再组装
图6  再组装PCV2 VLPs的稳定性
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