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Expression of the outer capsid protein, VP2, from a full length cDNA clone of genome segment 2 of bluetongue serotype 1 from South Africa, using both Sp6 and vaccinia expression systems and a comparison of the nucleic acid sequence of this segment with those of other serotypes



Expression of the outer capsid protein, VP2, from a full length cDNA clone of genome segment 2 of bluetongue serotype 1 from South Africa, using both Sp6 and vaccinia expression systems and a comparison of the nucleic acid sequence of this segment with those of other serotypes



Virus Research 15(3): 213-230



Genome segment 2 of bluetongue virus serotype 1 from South AfricA (BTV-1SA) was purified from a preparation of all ten dsRNA segments. This dsRNA was used as a template to make a full-length DNA copy of segment 2, which was then cloned into pUC19. The cDNA insert was transferred into a bacterial expression vector (pGEM; PROMEGA) and, by means of in vitro transcription and translation systems, used to synthesise a polypeptide of similar size to VP2 (as analyzed by PAGE). The cDNA insert was also transferred into a vaccinia virus vector using homologous recombination. The resulting recombinant virus when transfected into TK- cells produced a protein that co-migrated with VP2 of bluetongue virus. Immunoprecipitation of these polypeptides, synthesised by in vitro and in vivo techniques, using BTV-1SA antisera, confirmed that they were virus specific. Nucleotide sequence analysis of the cDNA demonstrated that genome segment 2 is 2940 base pairs in length. The positive sense (+ ve) RNA strand contains an open reading frame, coding for a polypeptide of 961 amino acids, which is flanked by 3' and 5' terminal non-coding regions of 37 and 17 nucleotides, respectively. Comparison with published data shows that genome segment 2 of BTV-1SA is identical in these characteristics to segment 2 of BTV-1 from Australia (BTV-1AUS) but differs from isolates of the five American serotypes of BTV (BTV-2, -10, -11, -13 and -17). However, there is a higher level of homology, in both the nucleotide and the amino acid sequence of genome segment 2 and protein VP2 respectively, between the two isolates of BTV-1 and the American isolate of BTV-2, than there is between BTV-2 and the other American serotypes. The significance of this similarity is discussed.

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Accession: 002105840

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PMID: 2160764

DOI: 10.1016/0168-1702(90)90029-b


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