Expression and immunogenicity of non-structural protein 8 of porcine epidemic diarrhea virus

Abstract

The non-structural protein (nsp) 8 of the porcine epidemic diarrhea virus (PEDV) is highly stable across different PEDV strains and plays an important role in PEDV virulence. In current study, nsp8 prokaryotic expression vectors were constructed based on parental vectors pMAL-c2x-maltose binding protein (MBP) and pET-28a (+). Subsequently, the optimization of expression conditions in Escherichia coli, including induced temperature, time and isopropyl β-D-thiogalactopyranoside concentration were performed to obtain a stable expression of MBP-nsp8 and nsp8. The nsp8 fused with MBP increased the water solubility of the expressed products. Target proteins were further purified from E. coli culture and their immunogenicities were evaluated in vivo by mice. The antibody titers of serum from nsp8 immunized mice were up to 1:7,750,000 when measured by indirect enzyme-linked immunosorbent assay; meanwhile, the mice immunized with MBP-nsp8 gave an antibody titer reaching 1:1,000,000. In all, the expression and purification system of PEDV nsp8 and MBP-nsp8 were successfully established in this work and a strong immune response was elicited in mice by both purified nsp8 and MBP-nsp8, providing a basis for the study of the structure and function of PEDV nsp8.

Keywords: Escherichia coli; Immunogenicity; Non-structural protein 8; Porcine epidemic diarrhea virus.

Fig. 1

Predictions for the porcine epidemic diarrhea virus non-structural protein (nsp) 8 hydrophobic domains, B cell antigenic epitopes and three-dimension (3D) structure. A) Protscale-based hydrophobic region prediction; B) BepiPred with a threshold value of 0.50 was used to predict the probable antigenic epitopes for B cells; C) The Swiss-model-predicted 3D structure of nsp8 marked with antigenic epitopes. After 90.00-degree rotation of the left panel image, the right panel image was produced

Fig. 2

Construction and verification of recombinant plasmid pMAL-c2x-maltose binding protein-non-structural protein (nsp) 8 and pET-28a (+)-nsp8. The position of nsp8 within the porcine epidemic diarrhea virus genome and construction strategy are shown in the schematic diagram

Fig. 3

The inserted gene fragment verification using 1.00% gel electrophoresis. A) Verification of the size of amplified target fragment of non-structural protein (nsp) 8; B) Double digestion of pMAL-c2x-maltose binding protein-nsp8 using EcoR I and Sal I; C) Double digestion of pET-28a (+)-nsp8 using EcoR I and Sal I. The red rectangles indicate nsp8

Fig. 4

Expression and purification of maltose binding protein (MBP)-non-structural protein (nsp) 8 and nsp8. A) Optimization of MBP-nsp8 expression conditions; B) Monitoring the nsp8 induction patterns throughout the course of 4 hr at 37.00 ˚C with various isopropyl β-D-thiogalactopyranoside (IPTG) supplementary; C) Monitoring of nsp8 induction patterns with 0.10 mM IPTG induction at 37.00 ˚C throughout a range of time durations; D) The nsp8 expression in pellet. M: Marker; Pre-I: Pre-induction; Post-I: Post-induction; SN: Supernatant; FT: Flow through

Fig. 5.

A) The maltose binding protein (MBP)-non-structural protein (nsp) 8 content in 40.00 mM imidazole; B) The nsp8 content in 40.00 mM imidazole. M: Marker; E1 - E14: Elution fraction from each number. The protein was eluted gradually at the concentration of 40.00 mmol mL^-1 imidazole

Fig 6.

A) The maltose binding protein (MBP)-non-structural protein (nsp) 8 was identified by MBP-tag antibody; B) The nsp8 was identified by His-tag antibody. The electrophoresis of each gel was performed using 12.00% polyacrylamide gels; C) The result of western blots of porcine epidemic diarrhea virus (PEDV)-infected Vero cells with anti-nsp8 mice serum (1:7,000). M: Marker; IB: Immunoblotting

Fig. 7

The antibody titers of maltose binding protein (MBP)-non-structural protein (nsp) 8 and nsp8 in the serum of immunized mice were tested by enzyme-linked immunosorbent assay at the given time periods by the immunoglobulin G antibodies

Fig. 8

A) The result of western blots of purified non-structural protein (nsp) 8 with anti-nsp8 mice serum that has been diluted (1:7,000); B) The result of western blots of purified maltose binding protein (MBP)-nsp8 with mice serum that has been diluted (1:7,000). Anti-phosphate-buffered saline (PBS) mice serum was considered as a negative control. M: Marker