Summary auto-generated
Researchers successfully expressed and purified a recombinant GB virus C (GBV-C) E2 envelope glycoprotein fragment (E2-315) in Chinese hamster ovary cells. The 315 amino acid protein was secreted, N-glycosylated, and purified using affinity chromatography to greater than 90% purity. The protein's N-terminal sequence and reactivity with antibodies confirmed proper expression. Importantly, the native, properly folded E2-315 protein was significantly more effective at detecting human antibodies against GBV-C in serum samples compared to denatured protein, indicating that conformational epitopes are critical for the immune response. Testing 28 human serum samples revealed 19 positive for E2 antibodies using native E2-315 ELISA, with most of these being negative for GBV-C RNA by RT-PCR, suggesting the presence of E2 antibodies may correlate with viral clearance. The results demonstrate the clinical utility of native glycosylated E2 protein for investigating GBV-C infection and epidemiology.
Key findings
- Recombinant GBV-C E2-315 protein was successfully expressed and secreted from CHO cells as a properly folded, N-glycosylated protein of 48-56 kDa
- Native E2-315 protein was significantly more effective than denatured E2 protein at detecting human antibodies to GBV-C in serum samples, indicating conformational epitopes are essential for immune recognition
- Human sera reactivity to E2 antibodies showed negative correlation with GBV-C RNA detection, suggesting E2 antibodies may be associated with viral clearance rather than active infection
- Native E2-315 ELISA detected 19 of 28 human serum samples as positive for E2 antibodies, indicating higher GBV-C prevalence than observed by RNA detection alone
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Abstract
A 315 amino acid recombinant segment of the GB virus C (GBV-C) E2 envelope glycoprotein (E2-315) was expressed and secreted from CHO cells. E2-315 was purified by affinity chromatography using a monoclonal antibody directed to a FLAG sequence genetically engineered onto the C terminus of the recombinant protein. The secreted protein had a molecular mass of 48-56 kDa and was shown to be N-glycosylated. Amino acid sequencing confirmed the expected N-terminal sequence. Purified E2-315 was used to develop an ELISA for detection of E2 antibodies in human sera. Antibodies to GBV-C E2 appeared to be directed toward conformational epitopes since human sera reactivity was detected in ELISA using native E2-315, but it was extremely weak or non- existent with denatured E2 protein. The use of an ELISA which can detect human GBV-C E2 antibodies will be important in further understanding of the clinical significance and epidemiology of GBV-C.