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Updated: Sep 20, 2026
Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins
Published on: April 17, 2020
Expression and glycosylation of rotavirus strain SA11 VP4 protein in a recombinant adenovirus
1Department of Molecular Biology, Institute of Medical Biology, CAMS & PUMC, Kunming 650118.
Objective:
Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain.
Methods:
A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal. The chimera gene was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co-transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation.
Results:
No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed product in recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosylation of VP4 protein.
Conclusion:
To modify the target gene might be an effective method to enhance the stability, antigenicity and immunogenicity of expressed protein.
