Prokaryotic expression and characterization of truncated mutant capsid protein of genotype IV hepatitis E virus.
- Author:
Yilan QIU
;
Junwen WU
;
Guo QIU
;
Sang LI
;
Ye LI
;
Shengzi LIU
;
Rushi LIU
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Monoclonal;
Antibodies, Neutralizing;
Blotting, Western;
Capsid Proteins;
biosynthesis;
Electrophoresis, Polyacrylamide Gel;
Enzyme-Linked Immunosorbent Assay;
Epitopes;
Genotype;
Hepatitis E virus;
Mice;
Mutant Proteins;
biosynthesis;
Open Reading Frames;
Recombinant Proteins;
biosynthesis
- From:
Chinese Journal of Biotechnology
2014;30(3):381-392
- CountryChina
- Language:Chinese
-
Abstract:
A truncated mutant of the Open Reading Frame 2 (ORF2, aa384-606) was amplified from cDNA of genotype IV hepatitis E virus (HEV) by polymerase chain reaction (PCR), subcloned to expression plasmid pTO-T7, and expressed in Escherichia coli. SDS-PAGE and Western blotting were used to detect and identify the recombinant protein, namely rP24. After washing of inclusion bodies, dissolving in denaturing agents, refoldeding by dialysis, ion exchange chromatography and gel chromatography, dynamic light scatter was used to study the hydrated radius of rP24. Western blotting was applied to detect the immunoreactivity of rP24, and mouse immunity test and indirect enzyme linked immunosorbent assay (ELISA) were applied to evaluate the immunogenicity and the detection rate of HEV positive and negative serum. SDS-PAGE and Western blotting show that rP24 was highly expressed in the form of inclusion bodies after induction, and had strong immunoreactivity to monoclonal antibody (McAb) 15B2. After a multi-step purification of rP24, Western blotting indicated that the purified rP24 also had strong immunoreactivity to neutralizing McAb 8C11 and HEV positive serum, suggesting that rP24 simulated the nature structure of HEV capsid protein. Dynamic light scatter demonstrated that the average hydration radius of purified rP24 was 7.48 nm. The mouse immunity test showed that the purified rP24 also had good immunogenicity, and the period of serum antibodies converted from negative to positive was very short, but the antibodies maintained more than 20 weeks. Indirect ELISA tests showed that the detection rate of was the same as anti-HEV-IgG diagnostic kit (Wan Tai corporation). Taken together, the rP24 simulated the neutralizing epitopes of natural HEV, and had strong immunoreactivity and immunogenicity. It provided a basis for the further investigation of the difference of infection mechanism between genotype I and genotype IV HEV.
