Characterization of genotype IV hepatitis E virus-like particles expressed in E.coli

Abstract

HEV (Hepatitis E virus) is an infectious disease transmitted between humans and animals, which poses a severe threat to the biological safety and property throughout the world. The disease is especially severe in patients with potential liver cirrhosis and women during pregnancy. There is no specific and thorough HEV treatment at present. The development of hepatitis E virus vaccine is vital to the prevention of viral hepatitis worldwide. Since HEV cannot grow adequately in vitro, vaccine developed by devitalized virus particles does not work. Exploration of HEV-like structures is essential for the development of functional vaccines against HEV infection. ORF2 encodes the structural proteins of HEV, some of which can automatically assemble into virus-like particles (VLP) in this experiment, the recombinant capsid protein p27 was expressed in E. coli and the VLP formed by p27 was used to immunize mice. The results showed that the VLP formed by recombinant P27 had similar particle size to that of HEV; the immune dose produced by p27 was positively correlated with the immune effect. Compared with other genetic engineering subunit vaccines, P27 protein has a better application prospect.

Keywords: HEV vaccine; Hepatitis E; The recombinant capsid protein p27; Virus-like particles.

Fig. 1

Expression, identification and purification of recombinant p27 in E. coliFig. 1A; Expression and identification of p27. A1: SDS-PAGE of expression of HEV p27, M: DNA molecular mass standard; 1: empty vector expression induced by IPTG; 2: B11-p27 induced expression; A2: Western Blot alaysis against murine monoclonal antibody 15B2 of recombinant p27, M: marker; 1: empty vector expression induced by IPTG; 2: B11-p27 induced expression; The arrow shows p27 Fig. 1B: Purification of recombinant protein p27. B1: SDS-PAGE analysis the expression forms of p27 protein, M: marker; 1: suspension; 2: deposit; 3: supernate; B2: SDS-PAGE of HEV p27 resolved in different concentration of urea, M: marker; 1–3: HEV p27 resolved in 2 M、4 M、8 M urea/Buffer I; B3: Western Blot of HEV p27 resolved in different concentration of urea, M: marker; 1–3: HEV p27 resolved in 2 M、4 M、8 M urea/Buffer I; Fig. 1C. Identification the antigenicity of renatured recombinant protein. C1.: SDS-PAGE of renatured recombinant protein, M: marker; 1: Denatured p24 protein (red arrow); 2: p24 protein (red triangle); 3: Denatured p27 protein (yellow arrow); 4: p27 protein (yellow triangle); C2: Western Blot of renatured recombinant protein which react with monoclonal antibody 8C11, M: marker; 1:Denatured p24 protein; 2: p24 protein: 3: Denatured p27 protein; 4: p27 protein. C3: SDS-PAGE of renatured recombinant protein, M: marker; 1:Denatured p27 protein; 2: p27 protein; 3:Denatured p24 protein; 4: p24 protein; C4: Western Blot of recombinant protein which react with positive serum, M: marker; 1:Denatured p27 protein; 2: p27 protein; 3:Denatured p24 protein; 4: p24 protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Ion exchange and molecular sieve chromatogram of p24 and p27 Protein. A1: Ion exchange chromatogram of p24 Protein; A2: Ion exchange chromatogram of p27 Protein. The X-axis represents the restain time. The Y-axis represents the voltage. The stronger the anion and the larger the molecular weight, the longer the restain time. B1: Molecular sieve chromatogram of p24 protein; B2: Molecular sieve chromatogram of p27 protein. The X-axis represents the restain time. The Y-axis represents the voltage.

Fig. 3

SDS-PAGE of p24 and p27 protein which purified by ion exchange chromatography and molecular sieve chromatography. A1: SDS-PAGE of purified p24 protein, M: marker; 1: p24 protein before Ion exchange chromatograpy (red arrow shows dimer); 2: p24 protein after Ion exchange chromatography (yellow arrow shows dimer); 3: p24 protein after molecular sieve chromatography (triangle shows dimer); A2: SDS-PAGE of purified p27 protein, M: marker; 1: p27 protein before Ion exchange chromatograpy (arrow shows polymer); 2: p27 protein after Ion exchange chromatography (yellow arrow shows polymer); 3: p27 protein after molecular sieve chromatography (triangle shows polymer). B: Native PAGE and Western blot of purified HEV p27. SDS-PAGE: 1: denatured p27 protein; 2: p27 protein; Western blot: 3: denatured p27 protein; 4: p27 protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Hydrated radius and Electron microscopy photo of purified p27 protein. A:The hydrated radius of purified p27 protein in solution. The vertical axis represents percentage intensity and the horizontal axis represents size. B1 The Electron microscopy photo of HEV p27 recombinant particle. A: Indicated Magnification: × 150,000: B2: Indicated Magnification: × 300,000.

Fig. 5

HEV p27 recombinant particle under atomic force microscopy. Fig. 5 A HEV p27 recombinant particle under atomic force microscopy A:Scanning Image of p27 in 4 μm, Fig. 5 B: three-dimensional image of p27 in 4 μm, Fig. 5 C: Scanning Image of p27 in 1.5 μm, Fig. 5 D: three-dimensional image of p27 in 1.5 μm.

Fig. 6

The seroconversion of mice serum after immunized with p27. Fig. 6 The seroconversion of mice serum after immunized with p27. The vertical axis shows the serum conversion rate. The horizontal axis show the immune time; n = 3.