Expression of caspid protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection

Abstract

To produce enterovirus 71 antigen for diagnostic purposes, the gene encoding the entire capsid protein VP1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and expressed in Escherichia coli as a poly-histidine fusion protein. Western blotting experiments with sera from patients with enterovirus 71 infection indicated that immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide VP1. According to these results, IgM anti-VP1 appeared in sera of patients with a symptomatic enterovirus 71 acute infection, whereas IgG anti-VP1 was present in sera of past infection. This finding suggests that detecting IgG and IgM immune responses against linear epitopes of recombinant VP1 is an effective means of determining the different phases of enterovirus 71 infection. In addition, sera containing coxsackie virus 16 (CA16) antibodies did not cross-react with the recombinant VP1 of enterovirus 71, despite the homology between VP1 proteins of both viruses. Comparison with reference PCR and neutralization assays showed these antibody tests to be appropriate for the serodiagnosis of enterovirus 71 infection.

Immunogenicity of a truncated enterovirus 71 VP1 protein fused to a Newcastle disease virus nucleocapsid protein fragment in mice

“…In the present study, purified N protein of NDV, and VP1 protein of EV71 were used to determine murine IgG responses, as measured by ELISA and Western blot analyses. The potential of the individual N (Makkay et al, 1999) and VP1 proteins (Shih et al, 2000) as diagnostic agents has been evaluated. In our study, a recombinant N-VP1 fusion protein, namely Nt-VP1t was used.…”

Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71

“…Total DNA-free RNA was extracted from the prototype EV71 2272-infected cells using TRIzol reagent-chloroform (GibcoBRL) extraction as described earlier [14]. The concentration of RNA was determined spectrophotometrically at 260/280 nm and quality as assessed by visualization on an ethidium stained agarose gel.…”

“…RT-PCR for cDNA synthesis and amplification was performed in a single step by adding RNA template and primers to RT-PCR beads (Amersham Pharmacia Biotech, Buckinghamshire, UK) containing MmuLV reverse transcriptase, RNase inhibitor, buffer, nucleotides, and taq polymerase. Two PCR oligodeoxynucleotide primers were synthesized as described earlier [14]; however, an NcoI and a HindIII restriction endonuclease site were introduced to allow ligation of the entire VP1 cDNA into pKK233-2, a prokaryotic expression vector with Salmonella-specific in vivo-regulated promoter [15] (Fig. 1A).…”

Development of polyclonal antisera to clone enterovirus 71 cellular receptor

“…It was shown that serum samples from patients infected with ENV71 recognise linear epitopes of a recombinant VP1, [19] indicating that these epitopes are immunogenic when a productive infection is progressing in vivo. The fi nding of our study is also in line with these reports.…”