GB virus C E2 glycoprotein: expression in CHO cells, purification and characterization

Area of Science:

• Virology
• Immunology
• Biochemistry

Background:

• GB virus C (GBV-C) is a human flavivirus.
• The E2 envelope glycoprotein is a key target for host immune responses.
• Understanding GBV-C antibody prevalence is important for clinical and epidemiological studies.

Purpose of the Study:

• To produce and purify a recombinant GBV-C E2 envelope glycoprotein fragment (E2-315).
• To develop an Enzyme-Linked Immunosorbent Assay (ELISA) for detecting antibodies against GBV-C E2 in human sera.
• To investigate the nature of epitopes recognized by GBV-C E2 antibodies.

Main Methods:

• Expression and secretion of a 315 amino acid recombinant GBV-C E2 glycoprotein (E2-315) from CHO cells.
• Purification of E2-315 using affinity chromatography with a FLAG-specific monoclonal antibody.
• Characterization of purified E2-315, including molecular mass determination and N-glycosylation analysis.
• Development and validation of an ELISA using native and denatured E2-315 to detect human antibodies.

Main Results:

• Recombinant E2-315 was successfully expressed, secreted, and purified.
• The purified E2-315 protein (48-56 kDa) was confirmed to be N-glycosylated.
• The developed ELISA detected antibodies to GBV-C E2 in human sera.
• Antibody reactivity was significantly higher with native E2-315 compared to denatured E2, indicating recognition of conformational epitopes.

Conclusions:

• A functional ELISA for detecting GBV-C E2 antibodies has been developed.
• GBV-C E2 antibodies primarily target conformational epitopes.
• This assay will aid in understanding the clinical significance and epidemiology of GBV-C infections.