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Cloning, Sequencing And Expression Of The Gene That Encodes The Major Neutralisation-specific Antigen Of African Horsesickness Virus Serotype 9.
Published 2000 · Medicine, Biology
A marked improvement in the efficiency of cloning the large double stranded RNA (dsRNA) genome segments of African horsesickness virus (AHSV) was achieved when the dsRNA polyadenylation step was carried out with undenatured rather than strand-separated dsRNA. It is a prerequisite to use dsRNA of very high purity because in the presence of even trace amounts of single stranded RNA, the dsRNA appears to be poorly polyadenylated as judged by its effectiveness as a template for oligo-dT-primed cDNA synthesis. The full-length VP2 gene of AHSV-9, cloned by this approach, was sequenced and it was found to show the highest percentage identity (60%) to VP2 of AHSV-6, providing an explanation of why these two serotypes show some cross protection. The VP2 protein was also expressed in Spodoptera frugiperda (Sf9) cells by means of a baculovirus recombinant. The yield of the expressed VP2 was high, but the protein was found to be largely insoluble. Nine smaller, truncated VP2 peptides were subsequently expressed in insect cells, but no significant improvement in solubility of the peptides, as compared to that of the full-sized protein, was observed. A western immunoblot analysis of the overlapping peptides indicated the presence of a strong linear epitope located within a large hydrophilic domain between amino acids 369 and 403.