Gene Modification, Expression and Immunogenicity of Rotavirus VP7

Abstract: Rotaviruses are the single most important cause of severe diarrhea in young children all over the world. VP7 is the major outer capsid and is a primary candidate for inclusion in a subunit or recombinant vaccine.Part of the VP7 gene containing all the three antigenic regions was expressed as a chimeric protein with glutathione S-transferase(GST) in E.coli. The chimeric protein, representing about 30% of the total protein of the recombinant-plasmid-carrying bacteria, reacted with polyclonal antibodies raised against whole virus. Immunization of sero-negative rabbits and mice with purified fusion-protein generated both virus-binding and neutralizing antibodies.To enhance the immunogenicity of VP7, the gene was engineered at the amino terminus by substitution of the influenza virus hemagglutinin(HA) signal sequence for the VP7 native signal sequence to direct VP7 into secretion. The secreted VP7(VP7s) was further modified into a cell-surface-anchored antigen(VP7tm) by the addition of the membrane anchor sequence from the influenza virus HA. The modifications were confirmed by DNA sequence analysis and gene expression in Hela cells.Expression vectors pcDNA3-VP7wt, pcDNA3-VP7s and pcDNA3-VP7tm bearing wild-type, secreted or membrane-anchored VP7 gene, respectively, were constructed and used as DNA vaccines. Intromuscular injections of mice with these constructs stimulated neutralizing antibodies. But neither the VP7s nor the VP7tm based DNA vaccine generated stronger VP7-specific IgG response than the VP7wt based vaccine did. Coadministration of recombinant VP7_(TR) with DNA vaccine induced significantly (P<0.05) greater serum Ig…
Key words: Rotavirus VP7; E.coli Expression; Gene modification; DNA immunization; Recombinant adenovirus

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