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Frontiers of Agricultural Science and Engineering

ISSN 2095-7505

ISSN 2095-977X(Online)

CN 10-1204/S

Postal Subscription Code 80-906

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Front. Agr. Sci. Eng.    2015, Vol. 2 Issue (3) : 230-236    https://doi.org/10.15302/J-FASE-2015071
RESEARCH ARTICLE
Efficient purification of cell culture-derived classical swine fever virus by ultrafiltration and size-exclusion chromatography
Ruining WANG1,Yubao ZHI2,Junqing GUO2,Qingmei LI2,Li WANG2,Jifei YANG2,Qianyue JIN2,Yinbiao WANG2,Yanyan YANG2,Guangxu XING2,Songlin QIAO2,Mengmeng ZHAO1,Ruiguang DENG2,Gaiping ZHANG1,*()
1. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China
2. Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
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Abstract

Large-scale production of cell culture-based classical swine fever virus (CSFV) vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium. Hence, we have developed an efficient method for purifying CSFV from cell-culture medium. Pure viral particles were obtained with two steps of tangential-flow filtration (TFF) and size-exclusion chromatography (SEC), and were compared with particles from ultracentrifugation by transmission electron microscopy (TEM), infectivity and recovery test, and real time fluorescent quantitative PCR (FQ-PCR). TFF concentrated the virus particles effectively with a retention rate of 98.5%, and 86.2% of viral particles were obtained from the ultrafiltration retentate through a Sepharose 4 F F column on a biological liquid chromatography system. CSFV purified by TFF-SEC or ultracentrifugation were both biologically active from 1.0×10−4.25 TCID50·mL−1 to 3.0×10−6.25 TCID50·mL−1, but the combination of TFF and SEC produced more pure virus particles than by ultracentrifugation alone. In addition, pure CSFV particles with the expected diameter of 40–60 nm were roughly spherical without any visible contamination. Mice immunized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultracentrifugation-purified CSFV (P<0.05). The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.
Keywords classical swine fever virus      virus purification      tangential-flow filtration      size-exclusion chromatography     
Corresponding Author(s): Gaiping ZHANG   
Just Accepted Date: 09 October 2015   Online First Date: 27 October 2015    Issue Date: 10 November 2015
 Cite this article:   
Ruining WANG,Yubao ZHI,Junqing GUO, et al. Efficient purification of cell culture-derived classical swine fever virus by ultrafiltration and size-exclusion chromatography[J]. Front. Agr. Sci. Eng. , 2015, 2(3): 230-236.
 URL:  
https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2015071
https://academic.hep.com.cn/fase/EN/Y2015/V2/I3/230
Fig.1  Schematic representation of tangential-flow filtration
Fig.2  Elution profile of size-exclusion chromatography and detection of CSFV by FQ-PCR. (a) The existence of CSFV in samples at various purification stages were monitored by FQ-PCR. 1, Virus stock; 2, ultrafiltration retentate; 3, ultrafiltration penetration; 4, peak 1; 5, peak 2; 6, ultracentrifugation-prepared virus; (b) TFF-concentrated CSFV was loaded onto a Sepharose 4 Fast Flow column (100 mL bed volume). Peak 1 consisted mainly of CSFV particles and peak 2 was contaminants from host cells or cell culture media.
Purification stageTotal volume/mLParticles numbers/mLVirus recovery a /%Virus titer /TCID50·mL−1
Virus stock5001.36×10101005.5×10−4.25
UF rententate203.35×101198.53.4×10−6.35
UF permeate485N/AN/AN/A
Flow-through peak 1202.89×101186.23.0×10−6.25
Flow-through peak 2205.07×102N/A1.0×10−1
Ultracentrifugation202.56×101185.84.09×10−5
Tab.1  Monitoring of virus particles at various stages of purification
Fig.3  Analysis of the purified CSFV by TFF-SEC by SDS-PAGE (a) and immunoblotting (b). (a) SDS-PAGE: lane 1, peak 1 sample; lane 2, peak 2 sample; M, pre-stained protein molecular marker; (b) immunoblotting: lane 3, peak 2 sample; lane 4, peak 1 sample.
Fig.4  Immunofluorescence assay. (a) Monolayer of PK-15 cells infected with TFF-SEC-purified CSFV; (b) monolayer of PK-15 cells infected with ultracentrifugation-prepared CSFV. Mock-uninfected PK-15 cells were used as negative control.
Fig.5  Electron microscopy of the purified CSFV particles. (a) CSFV particles purified by TFF-SEC are spherical with a diameter of 50 nm; (b) CSFV prepared by ultracentrifugation show few visible intact virions and a significant amount of contaminating substances is evident as the gray background. Primary magnification of 100000× and 300000×.
Fig.6  Comparison of antibody responses. Antibody levels from immunization with CSFV purified from TFF-SEC (group A) and ultracentrifugation (group B) were tested with the strip test and compared. Mice immunized with PBS in group C did not produce any antibody against CSFV.
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