Cloning, sequencing and expression of the gene that encodes the major neutralisation-specific antigen of African horsesickness virus serotype 9

A marked improvement in the efficiency of cloning the large double stranded RNA (dsRNA) genome segments of African horsesickness virus (AHSV) was achi eved when the dsRNA polyadenylation step was carried out with undenatured r ather than strand-separated dsRNA. It is a prerequisite to use dsRNA of ver y high purity because in the presence of even trace amounts of single stran ded RNA, the dsRNA appears to be poorly polyadenylated as judged by its eff ectiveness as a template for oligo-dT-primed cDNA synthesis. The full-lengt h VP2 gene of AHSV-9, cloned by this approach, was sequenced and it was fou nd to show the highest percentage identity (60%) to VP2 of AHSV-6, providin g an explanation of why these two serotypes show some cross protection. The VP2 protein was also expressed in Spodoptera frugiperda (Sf9) cells by mea ns of a baculovirus recombinant. The yield of the expressed VP2 was high, b ut the protein was found to be largely insoluble. Nine smaller, truncated V P2 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 overlapp ing peptides indicated the presence of a strong linear epitope located with in a large hydrophilic domain between amino acids 369 and 403. (C) 2000 Els evier Science B.V. All rights reserved.