Cloning, expression and purification of envelope proteins E1 and E2 of western equine encephalitis virus and potential use of them as antigens in immunoassays.

The researchers cloned and expressed the E1 and E2 proteins of the Western Equine Encephalitis Virus (WEEV), purified them and tested their potential as antigens in immunoassays for detection of anti-WEEV antibodies.

Cloning and Expression of Recombinant Envelope Proteins

• The genes that encode the envelope proteins E1 and E2 of the WEEV were cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. These vectors are often used to facilitate and study the expression of specific proteins within the host organism.
• The recombinant proteins, tagged with a 6xHis, were expressed within bacteria. This method is typically used to help visually identify and purify overexpressed target proteins from other host proteins.

Purification of Recombinant Proteins

• The expressed proteins were formed into inclusion bodies, solubilized with 8M urea (a compound commonly used to dissolve proteins) and then purified using a technique known as immobilized metal ion affinity chromatography. This method uses the affinity of proteins, specifically the 6xHis-tagged WEEV E1 and E2, for certain metal ions to selectively isolate the target protein.
• Purification was confirmed by identifying the target proteins as 50kDa bands in a SDS-PAGE gel. This result was consistent with the expected sizes of the WEEV E1 and E2 proteins.
• The proteins were then refolded using an arginine system to help maintain their natural, functional conformation.

Potential use as Antigens in Immunoassays

• The purified proteins were then tested as potential antigens in immunoassays, specifically enzyme-linked immunosorbent assays (ELISAs), to detect anti-WEEV antibodies.
• When confronted with anti-WEEV antibodies from mice infected with WEEV, these proteins showed a positive, dose-dependent reaction, pointing toward their potential as reliable antigens for the detection of WEEV infection.
• Conversely, when knowing the genetic background, antibodies against the Venezuelan Equine Encephalitis Virus, which is very similar to WEEV, did not bind significantly to the recombinant WEEV E1 and E2 proteins. This is an important finding that confirms that the recombinant proteins are not cross-reactive and thus are specific to WEEV.
• The research suggests that these recombinant WEEV E1 and E2 proteins could act as valuable antigens in immunoassays, and could replace the need for the more laborious, costly, and potentially hazardous cell-culture derived viral antigens currently used.