Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

The researchers have successfully cloned the full-length horse interferon-gamma (eIFN-gamma) cDNA and inserted it into a baculovirus vector. This led to the production of biologically active recombinant eIFN-gamma proteins in two different systems: insect cells and silkworm larvae. These proteins were shown to be effective against the vesicular stomatitis virus and induced the production of MHC class II antigen in equine kidney cells.

Recombinant eIFN-gamma cloning and production

• The researchers first cloned the full-length eIFN-gamma cDNA, including the secretion signal peptide coding region, from horses. This eIFN-gamma is an important cytokine that is involved in immune responses.
• This cloned cDNA was then incorporated into a baculovirus transfer vector named pAcYM1. Baculoviruses are commonly used in biotechnology for transferring genes into various cells.
• The vector, carrying the horse eIFN-gamma cDNA, was then integrated into two different virus DNAs: Autographa californica nuclear polyhedrosis virus DNA and a hybrid nuclear polyhedrosis virus DNA. This process was done via co-transfection into Spodoptera frugiperda cells.
• The recombinant baculoviruses produced (AcEIFN-gamma and HyEIFN-gamma) contained the desired eIFN-gamma gene.

Extraction and Testing of the Recombinant eIFN-gamma

• The eIFN-gamma was produced in the culture fluid of AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line (BTI TN 5B1-4) and in the hemolymph (blood equivalent) of HyEIFN-gamma infected silkworm larvae.
• These recombinant interferon-gamma forms (reIFN-gamma) were shown to be proteins weighing between 14 to 20 kiloDaltons. They were also confirmed to be glycosylated, which means sugar groups were attached to the protein, a common post-translational modification in proteins.
• Both reIFN-gamma proteins were shown to be biologically active, exhibiting high activity levels against the vesicular stomatitis virus by reducing its cytopathic effects. They also induced the expression of MHC class II antigen in equine fetal kidney-78 cell line, meaning they could stimulate immune responses.

These findings suggest that the baculovirus-insect cell and baculovirus-silkworm larvae systems can be employed as efficient platforms for the production of fully functional and biologically active eIFN-gamma proteins for research.