Prokaryotic expression and bioinformatics analysis of Nsp5 gene of porcine epidemic diarrhea virus

doi:10.3969/j.issn.1004-1524.2019.04.04

›› 2019, Vol. 31 ›› Issue (4): 532-538.DOI: 10.3969/j.issn.1004-1524.2019.04.04

• Animal Science • Previous Articles Next Articles

Prokaryotic expression and bioinformatics analysis of Nsp5 gene of porcine epidemic diarrhea virus

LIU Zhengkui¹, WU Yuan², CHEN Lin¹, WANG Lei¹, MU Hongye¹, ZHU Xuhang¹, WANG Xiaodu^1,*

1. College of Animal Science and Technology, Zhejiang A & F University, Hangzhou 311300,China;
2. School of Agricultural and Biological Engineer, Jinhua Polytechnic, Jinhua 321007,China

Received:2018-10-05 Online:2019-04-25 Published:2019-04-19

Abstract

Abstract: Porcine epidemic diarrhea virus (PEDV) nonstructural protein 5 (Nsp5) gene was expressed by the prokaryotic expression system and the structure of the encoded protein was predicted by bioinformatics software for understanding the pathogenecity mechanism of PEDV. In this study, the Nsp5 gene of PEDV/LY/2014/04 strain was subcloned into prokaryotic expression vector pET-28a, and the recombinant plasmid was identified by polymerase chain reaction (PCR) and restriction enzyme digestion. The recombinant plasmid pET-28a(+)-Nsp5 was transformed into competent cells of Escherichia coli BL21(DE3). The expression and purification effect of Nsp5 were detected by SDS-PAGE. The Vector NTI Advance, online software was utilized to predict and analyze the amino acid composition, epitope, secondary and tertiary structure of Nsp5 protein. The results showed that the recombinant plasmid pET-28a(+)-Nsp5 was successfully constructed, and approximately 22 ku recombinant protein was expressed and mainly existed in the form of inclusion bodies. High-purity recombinant protein was obtained after purifying by His band Ni+. The Nsp5 protein consisted of a polypeptide with 196 amino acid residues. The theoretical molecular mass of the slightly alkaline recombinant protein was 21 820.07 u, and the theoretical isoelectric point(pI) was 8.734. The secondary structure was composed of 55.61% α-helix(h), 7.65% β-fold(t), 20.92% irregular curl(c) and 15.82% extended chain(e). In the tertiary structure of the Nsp5 protein, the middle segment skeleton is mainly formed by α-helix, and a plurality of complex secondary structures at the C-end constituted the enzyme active center of the protein. The prediction of B cell epitope indicated that there were 15 potential B cell dominant epitope. This study provided data support for studying on the biological function of the porcine epidemic diarrhea virus Nsp5 protein.

Key words: porcine epidemic diarrhea virus, Nsp5 gene, prokaryotic expression, bioinformatics analysis

CLC Number:

S855.3

LIU Zhengkui, WU Yuan, CHEN Lin, WANG Lei, MU Hongye, ZHU Xuhang, WANG Xiaodu. Prokaryotic expression and bioinformatics analysis of Nsp5 gene of porcine epidemic diarrhea virus[J]. , 2019, 31(4): 532-538.

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Prokaryotic expression and bioinformatics analysis of Nsp5 gene of porcine epidemic diarrhea virus

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