Efficient Assembly of Virus-like Particles of Porcine Circovirus Type 2

doi:10.13523/j.cb.2006045

China Biotechnology

2020, Vol. 40

Issue (11): 35-42 DOI: 10.13523/j.cb.2006045

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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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

Received: 04 June 2020 Published: 11 December 2020

ZTFLH:

Q819

Corresponding Authors: Wen-qiang PANG,Ke-gong TIAN E-mail: pangwq123@hotmail.com;vetvac@126.com

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	Yan-wei WANG
	Peng-hao LI
	Yan-yu LIANG
	Yang GUAN
	Wen-qiang PANG
	Ke-gong TIAN

Cite this article:

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.

URL:

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

Fig.1 Purification and self-assembled VLPs of PCV2 rCap protein (a) Analysis of purified PCV2 rCap by SDS-PAGE M: Protein marker; 1: Supernatant of lysates; 2: Crude protein after ammonium sulfate precipitation; 3: Protein eluted by 500mmol/L NaCl (b) Analysis of purified PCV2 rCap by TEM (b:300 000×)

Fig.2 Stability of self-assembled PCV2 VLPs (a) Analysis of PCV2 VLPs by DLS in 500mmol/L or 150mmol/L NaCl (b)Nucleic acid test M: DL 5 000bp marker; 1: 500mmol/L NaCl; 2: 150mmol/L NaCl

Fig.3 Disassembly of PCV2 VLPs (a) Change of size with urea concentration (b) Change of size with pH (c) Change of Zeta potential with pH

Fig.4 Purification of PCV2 rCap after disassembly (a) Result of chromatography (b) Analysis by SDS-PAGE M: Protein marker; 1: Supernatant of lysates; 2: Crude protein after ammonium sulfate precipitation; 3: Protein eluted by 300mmol/L NaCl; 4: Protein eluted by 500mmol/L NaCl; 5: Protein eluted by 1mol/L NaCl (c) Analysis by nucleic acid test M: DL 5 000bp marker; 1: Protein eluted by 500mmol/L NaCl; 2: Protein eluted by 1mol/L NaCl

Table 1 Recovery of PCV2 rCap protein during purification

Fig.5 Reassembly of PCV2 VLPs (a) Change of Zeta potential with pH (b) Analysis by DLS (c) Analysis by TEM (300 000×) (d) Analysis by HPSEC

Fig.6 Stability of reassembled PCV2 VLPs (a) Analysis of reassembled PCV2 VLPs by DLS (b) Analysis of reassembled PCV2 VLPs by TEM (b:300 000×)


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