Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form.
The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus-cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26 Delta C), followed by purification by NTA-Ni(2+) agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies.
Royuela
Enrique
E
Centro Nacional de Microbiología (CNM), Instituto de Salud Carlos III (ISCIII), 28220 Madrid, Spain. eroyuela@isciii.es
Sánchez-Fauquier
Alicia
A
D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology / Q000378:metabolism; D000956:Antigens, Viral / Q000276:immunology* / Q000378:metabolism; D019350:Astroviridae Infections / Q000276:immunology / Q000821:virology; D036022:Capsid Proteins / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000276:immunology / Q000821:virology; D008253:Macaca mulatta; D001257:Mamastrovirus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism
D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D000939:Epitopes; D011994:Recombinant Proteins