Production of soluble truncated spike protein of porcine epidemic diarrhea virus from inclusion bodies of Escherichia coli through refolding
| Autor: | Da-Chuan Piao, Chong-Su Cho, Yun-Jaie Choi, Sang-Kee Kang, Zhong-Shan Hong, Yoonseok Lee, Jin-Duck Bok |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Swine Protein subunit 030106 microbiology medicine.disease_cause Subunit vaccine IBs inclusion bodies Inclusion bodies Article IPTG isopropyl d-1-thiogalactopyranoside PEDV porcine epidemic diarrhea virus 03 medical and health sciences Mice Chlorocebus aethiops medicine Solubilization Animals Refolding Escherichia coli CD circular dichroism Vero Cells Antiserum Inclusion Bodies biology Chemistry Immunogenicity Porcine epidemic diarrhea virus PEDV S protein Viral Vaccines biology.organism_classification HRP horseradish peroxide Virology S1 domain TBS Tris-buffered saline Titer OD optical density 030104 developmental biology Solubility DTT dithiothreitol Spike Glycoprotein Coronavirus Immunization Biotechnology |
| Zdroj: | Protein Expression and Purification |
| ISSN: | 1096-0279 1046-5928 |
| Popis: | The emergence of highly pathogenic variant porcine epidemic diarrhea virus (PEDV) strains, from 2013 to 2014, in North American and Asian countries have greatly threatened global swine industry. Therefore, development of effective vaccines against PEDV variant strains is urgently needed. Recently, it has been reported that the N-terminal domain (NTD) of S1 domain of PEDV spike protein is responsible for binding to the 5-N-acetylneuraminic acid (Neu5Ac), a possible sugar co-receptor. Therefore, the NTD of S1 domain could be an attractive target for the development of subunit vaccines. In this study, the NTD spanning amino acid residues 25-229 (S25-229) of S1 domain of PEDV variant strain was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies (IBs). S25-229 IBs were solubilized in 20 mM sodium acetate (pH 4.5) buffer containing 8 M urea and 1 mM dithiothreitol with 95% yield. Solubilized S25-229 IBs were refolded by 10-fold flash dilution and purified by one-step cation exchange chromatography with >95% purity and 20% yield. The CD spectrum of S25-229 showed the characteristic pattern of alpha helical structure. In an indirect ELISA, purified S25-229 showed strong reactivity with mouse anti-PEDV sera. In addition, immunization of mice with 20 μg of purified S25-229 elicited highly potent serum IgG titers. Finally, mouse antisera against S25-229 showed immune reactivity with native PEDV S protein in an immunofluorescence assay. These results suggest that purified S25-229 may have potential to be used as a subunit vaccine against PEDV variant strains. Highlights • Expression of truncated S25-229 of PEDV variant strain in Escherichia coli BL21 (DE3) resulted in inclusion body formation. • Soluble S25-229 was recovered by solubilization and refolding, followed by one-step cation exchange chromatography. • The CD spectrum of S25-229 showed the characteristic pattern of alpha helical structure. • The S25-229 showed immunogenicity and elicited strong humoral immune response in mice. |
| Databáze: | OpenAIRE |
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