水貂肠炎病毒VP2基因原核表达及病毒样颗粒制备

doi:10.16431/j.cnki.1671-7236.2021.02.004

Abstract

Abstract: This study was aimed to produce soluble protein of Mink enteritis parvovirus (MEV) capsid protein VP2 and to obtain virus-like particles (VLPs) of MEV.The MEV VP2 gene was optimized,synthesized and cloned into prokaryotic expression vector pET-30a(+).The recombinant vector pET-30a-VP2 was expressed by co-transformation with pTf16 into ER2566,and was induced at different temperatures,L-arabinose concentrations and IPTG concentrations,and analyzed by SDS-PAGE and Western blotting.VP2 was purified by ammonium sulfate precipitation combined with sucrose density gradient centrifugation,then identified by SDS-PAGE.Under the different assembled conditions,the purified protein was self-assemble into VLPs with the removal of sucrose.VLPs were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM).Minks were immunized with homemade MEV VLPs vaccine to identify their immunogenicity.The results showed that recombinant VP2 protein was highly expressed in the supernatant of E.coli after induction with 2 g/L L-arabinose and 0.2 mmol/L IPTG at 25 ℃ for 16 h.SDS-PAGE showed that the fusion protein was about 65 ku.Western blotting confirmed that VP2 had good antigen specificity.VP2 was purified by ammonium sulfate precipitation combined with sucrose density gradient centrifugation and degree of purity could reach more than 90%.DLS and TEM results indicated that VLPs had similar size,shape and hemagglutination (1:2¹⁴) with the authentic virus capsid at 150 mmol/L NaCl,pH 8.0.Immunization with VLPs vaccine could induce high-titer hemagglutination inhibition antibody (the highest was 1:2¹¹) in minks after 21 days,indicating that the VLP vaccine had good immunogenicity and could effectively prevent MEV.VLPs with high immunogenicity could be obtained by expression of VP2 with pTf16 in prokaryotic expression system,which laid a foundation for further research on MEV VLPs vaccine.

Key words: VP2; prokaryotic expression; pTf16; virus-like particles

CLC Number:

S855.3

WU Mingjie, XIA Linya, WANG Lei, ZHANG Ning, LUO Guoliang, YIN Yuhe, WU Congmei. Prokaryotic Expression of Mink Enteritis Parvovirus VP2 Gene and Assembly of Virus-like Particles[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 433-442.

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Prokaryotic Expression of Mink Enteritis Parvovirus VP2 Gene and Assembly of Virus-like Particles

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