Generation of soluble, immunoreactive recombinant JEV E protein through a simplified inclusion body extraction and refolding approach in vitro

Abstract

Japanese encephalitis (JE) is a mosquito-borne infectious disease caused by the Japanese encephalitis virus (JEV). There is currently no effective treatment for JE, and all approved Japanese encephalitis vaccine products originated from the JEV genotype III (GIII). In recent years, JEV genotype I (GI) has gradually replaced GIII as the dominant genotype, and a new symptom of peripheral nerve injury (PNI) caused by JEV NX1889 strain has attracted wide attention, in which JEV envelope (E) protein may be involved in early peripheral nerve injury. Here, the envelope (E) gene of JEV GIb, NX1889 strain was cloned into pET-32a (+) plasmid, and then the E protein was successfully expressed as the high-yield inclusion body form in Escherichia coli (E. coli) BL21 (DE3). Subsequently, we successfully developed a simplified approach to generate soluble JEV E protein by solubilization, and a stepwise dialysis refolding approach with the introduction of a redox system. The refolding recombinant E protein was identified by anti-JEV-E polyclonal antibody and the serum anti-E titer of mice immunized with refolded E protein was higher than 1: 409,600, which proves it has highly immunoreactive. Moreover, the recombinant E protein could recognize antibodies in samples of multiple species and types, such as mice, rabbits, and humans. The approach described above is economical and does not require complex eukaryotic expression systems to produce immunoreactive JEV E protein, which may be of great significance for the diagnosis and pathogenesis of JEV GI strain.

Keywords

• Japanese encephalitis virus (JEV)
• NX1889 strain
• Envelop (E)
• Inclusion body
• E. coli expression system