Burroughs J N, O'Hara R S, Smale C J, Hamblin C, Walton A, Armstrong R, Mertens P P
AFRC Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, U.K.
J Gen Virol. 1994 Aug;75 ( Pt 8):1849-57. doi: 10.1099/0022-1317-75-8-1849.
Methods were developed for the purification, at high yield, of four different particle types of African horsesickness virus serotype 9 (AHSV-9). These products included virus particles purified on CsCl gradients which contain proteins apparently directly comparable to those of bluetongue virus (VP1 to VP7); virus particles purified on sucrose gradients which also contain, as a variable component, protein NS2; infectious subviral particles (ISVPs), containing chymotrypsin cleavage products of VP2; and cores, obtained by treating purified ISVPs with 1 M-MgCl2 to remove the components of the outer capsid layer (VP5 and VP2 cleavage products). Additional protein bands migrating with apparent M(r)s lower than that of VP5 were detected during SDS-PAGE analysis of virus particles. These appear to be conformational variants of VP5 and are identified as VP5' and VP5". BHK-21 cells infected with this strain of AHSV-9 produce large quantities of flat, usually hexagonal crystals of VP7, a major group antigen and core protein; these were also purified. Either 20 mg of virus particles, 20 mg of ISVPs or 10 mg of cores as well as 20 mg of VP7 crystals could be purified from approximately 8 x 10(9) infected cells. None of the preparations of particles or crystals showed any detectable contamination with BHK-21 cell proteins or antigens, as determined by SDS-PAGE or indirect ELISA. Virus particle and ISVP preparations had similar specific infectivities for BHK-21 cells (approximately 1 x 10(9) TCID50/A260 unit) but the infectivity of cores was approximately 10(5)-fold lower.
已开发出多种方法，可高产量地纯化非洲马瘟病毒9型（AHSV-9）的四种不同颗粒类型。这些产物包括在氯化铯梯度上纯化的病毒颗粒，其所含蛋白质显然与蓝舌病毒的蛋白质（VP1至VP7）直接可比；在蔗糖梯度上纯化的病毒颗粒，其中还含有可变成分蛋白质NS2；感染性亚病毒颗粒（ISVP），含有VP2的胰凝乳蛋白酶裂解产物；以及核心颗粒，通过用1 M氯化镁处理纯化的ISVP以去除外衣壳层的成分（VP5和VP2裂解产物）而获得。在病毒颗粒的SDS-PAGE分析过程中，检测到迁移的额外蛋白带，其表观分子量低于VP5。这些似乎是VP5的构象变体，被鉴定为VP5'和VP5"。感染该AHSV-9毒株的BHK-21细胞会产生大量扁平的、通常为六边形的VP7晶体，VP7是一种主要的群抗原和核心蛋白；这些晶体也被纯化。从大约8×10⁹个感染细胞中可纯化出20 mg病毒颗粒、20 mg ISVP或10 mg核心颗粒以及20 mg VP7晶体。通过SDS-PAGE或间接ELISA测定，颗粒或晶体的所有制剂均未显示出任何可检测到的BHK-21细胞蛋白质或抗原污染。病毒颗粒和ISVP制剂对BHK-21细胞具有相似的比感染性（约1×10⁹ TCID₅₀/A₂₆₀单位），但核心颗粒的感染性约低10⁵倍。
