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In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection
Published on: November 27, 2014
[Immunochemical properties of West Nile virus protein prM and protein M C-end]
Abstract:
Complementary DNA fragments (nucleotides 466-966 and 878-1088) encoding prM protein and polypeptide M31-75-E1-30 of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were obtained and cloned. Recombinant polypeptides prM and M3175-E1-30 having amino acid sequences corresponding to the cloned cDNA fragments were purified by affinity chromatography. According to ELISA and Western blotting prM protein interacted with polyclonal antibodies against WNV. This is indicative the immunochemical similarity of WNV recombinant and native protein prM. 6 types of species-specific monoclonal antibodies (MAbs) raised against recombinant polypeptide prM recognized at least four epitopes within recombinant polypeptides prM and M31-75-E1-30. MAbs 7D11 were active in the virus - neutralization assay. Analysis of interaction of the MAbs with recombinant polypeptides prM, M31-75-EI-30, E1-180, E260-466 revealed cross-reactive epitopes within 260-466 amino acid residues (aa) of WNV protein E, 31-75 aa of polypeptide M31-75-E1-30 and protein prM. Proposed spatial model of proteins E and M C-end fragments shown similarity of their three-dimensional structures confirming results of immunochemical assay. Neutralization of viral infectivity by MAbs 7D11 raised against epitope within 31-75 aa t of protein M is evidence of important function of C-end region in the process of flaviviral penetration into host cell.
Insights
Researchers cloned West Nile virus (WNV) prM protein and M31-75-E1-30 fragments. Monoclonal antibodies identified epitopes, with 7D11 neutralizing WNV, highlighting the C-end region
Area of Science:
- * Virology
- * Molecular Biology
- * Immunology
Context:
- * West Nile virus (WNV) poses a significant public health threat.
- * Understanding WNV protein structure and function is crucial for developing antiviral strategies.
- * Recombinant protein expression and antibody development are key tools in virological research.
Purpose:
- * To clone and express complementary DNA fragments encoding WNV prM protein and a specific M polypeptide.
- * To generate and characterize recombinant prM and M31-75-E1-30 polypeptides.
- * To investigate the immunochemical properties and epitope mapping of WNV proteins using monoclonal antibodies.
Summary:
- * Complementary DNA fragments encoding West Nile virus (WNV) prM protein and M31-75-E1-30 were cloned and expressed as recombinant polypeptides.
- * Purified recombinant proteins were used to raise and test monoclonal antibodies (MAbs).
- * Six species-specific MAbs recognized at least four epitopes, with MAb 7D11 demonstrating WNV neutralization activity, indicating the importance of the C-terminal region in viral entry.
Impact:
- * The study confirms the immunochemical similarity between recombinant and native WNV prM protein.
- * Identified cross-reactive epitopes within WNV proteins E and M, providing insights into viral structure.
- * Neutralization of WNV by MAb 7D11 suggests a critical role for the C-terminal region of protein M in flavivirus cell entry, offering potential therapeutic targets.
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The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.