[Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay].

The research has successfully utilized a major antigenic domain of equine arteritis virus (EAV) – the GL protein, designing it for prokaryotic expression. Post this, an indirect ELISA assay was developed based on this artificially expressed protein. Initial testing proved promising, showing strong concordance with other testing methods and indicating a potential clinical application in detecting EAV antibodies.

Methodology and Results

• The researchers designed primers analysing the antigenic components of the EAV’s GL protein.
• The gene fragment that coded for this high antigenic domain was amplified from the EAV genome.
• This cloned gene was subsequently digested using restriction enzymes BamH I and Xho I.
• Post-digestion, the cloned gene was integrated into the pET-32a vector.
• To ensure the construct, dubbed pET-GL1, was successfully expressed, it was transformed into the BL21(DE3) host cell.
• Expression was optimized through the modulation of cultivation temperature and concentration of IPTG (Isopropyl β-D-1-thiogalactopyranoside), an inducing agent for genes under the control of the lac operator.
• The desired protein was highly expressed and recombinant GL1 protein exhibited high reactiongenicity, verified through Western blotting.

Purification and ELISA Assay Establishment

• The recombinant GL1 protein was purified using the His* Bind resin protein purification technique.
• An indirect ELISA (enzyme-linked immunosorbent assay) was established, the purified GL1 protein was used to coat the wells of the assay plate.
• Optimal concentration and dilution factors for the assay were identified as 9.65 micrograms/mL and 1:80 ratio respectively.
• Thresholds for positive and negative viral presence measured using optical density (OD) were established.

Validation of the ELISA Assay

• The established ELISA assay was compared with the micro-virus neutralization test, producing an agreement of 94.1%, indicating its effectiveness.
• Subsequent testing with 900 horse sera samples and comparison with the INGEZIM ELISA kit on 180 samples showed a matching ratio of 95.6%, further validating the developed assay.