Production of soluble truncated spike protein of porcine epidemic diarrhea virus from inclusion bodies of Escherichia coli through refolding.
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.
Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea.
Da-Chuan
DC
Piao
Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun, 232-916, Republic of Korea.
Yoon-Seok
YS
Lee
Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun, 232-916, Republic of Korea.
Jin-Duck
JD
Bok
Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea.
Chong-Su
CS
Cho
Department of Animal Science, Tianjin Agricultural University, Tianjin, 300-384, People's Republic of China.
Zhong-Shan
ZS
Hong
Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun, 232-916, Republic of Korea. Electronic address: kangsk01@snu.ac.kr.
Sang-Kee
SK
Kang
Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea; Department of Animal Science, Tianjin Agricultural University, Tianjin, 300-384, People's Republic of China. Electronic address: cyjcow@snu.ac.kr.
Yun-Jaie
YJ
Choi
D000818:Animals; D002522:Chlorocebus aethiops; D007114:Immunization; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D053485:Porcine epidemic diarrhea virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012995:Solubility; D064370:Spike Glycoprotein, Coronavirus / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013552:Swine; D014709:Vero Cells; D014765:Viral Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D064370:Spike Glycoprotein, Coronavirus; D014765:Viral Vaccines
Immunogenicity; Inclusion bodies; PEDV S protein; Refolding; Solubilization; Subunit vaccine