Gordon J, Mohandas A, Wilton S, Dales S
Department of Microbiology and Immunology, University of Western Ontario, London, Canada.
Virology. 1991 Apr;181(2):671-86. doi: 10.1016/0042-6822(91)90901-m.
Polypeptides of the vaccinia virus envelope exposed on the surface were identified by means of sulfo-N-hydroxysuccinimidobiotin as a surface tag. Among surface expressed polypeptides is the 35-kDa antigen, previously designated Ag35. Both monoclonal (mAb) and monospecific affinity pure antibodies directed against Ag35 neutralized vaccinia infectiousness, indicating that this prominent surface antigen has a function during early virus-host cell interactions. The binding of several monoclonal antibodies to various regions of Ag35 was tested by reacting CNBr fragments, derived from the polypeptide, employing Western blotting. All mAbs tested reacted with the same region of Ag35. Estimation of the molecular weights (MW), based on migration of the CNBr peptides in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that those partial digestion products which contained a proline-rich 99 amino acid limit digest fragment were present at a position approximately 12.5 kDa larger than that predicted from the DNA sequence. By contrast, partial and limit digest products lacking the proline-rich fragment migrated to the MW position expected from the length of the DNA sequence. This observation demonstrates that departure from a predicted 22.3 kDa to an anomalous MW of Ag35 is conferred by the proline-rich peptide. The surface location of Ag35 was confirmed by immune electron microscopy. In a competition test the binding specificity of mAb and affinity-purified antibodies at the surface of virions could be demonstrated. Evidence for an association of Ag35 with the virus envelope at various stages during biogenesis of vaccinia was obtained by immune electron microscopy of whole mounts and thin sections. Presence of Ag35 as an early component of immature and mature virions, probably residing in the bilayer membrane structure was detected. A distinction can, therefore, be made between Ag35 and several other vaccinia envelope polypeptides which are synthesized as late functions and added during late stages of envelope assembly.
通过磺基 - N - 羟基琥珀酰亚胺生物素作为表面标记物，鉴定出痘苗病毒包膜表面暴露的多肽。在表面表达的多肽中，有35 kDa抗原，先前称为Ag35。针对Ag35的单克隆抗体（mAb）和单特异性亲和纯抗体均能中和痘苗病毒的感染性，表明这种突出的表面抗原在病毒 - 宿主细胞早期相互作用中发挥作用。通过蛋白质印迹法，使来源于该多肽的溴化氰片段反应，测试了几种单克隆抗体与Ag35不同区域的结合情况。所有测试的单克隆抗体都与Ag35的同一区域反应。基于溴化氰肽在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中的迁移情况对分子量（MW）进行估计，结果显示，那些包含富含脯氨酸的99个氨基酸的极限消化片段的部分消化产物，其位置比根据DNA序列预测的位置大约大12.5 kDa。相比之下，缺乏富含脯氨酸片段的部分消化产物和极限消化产物迁移到了根据DNA序列长度预期的分子量位置。这一观察结果表明，富含脯氨酸的肽赋予了Ag35从预测的22.3 kDa偏离到异常分子量的特性。免疫电子显微镜证实了Ag35的表面定位。在竞争试验中，可以证明单克隆抗体和亲和纯化抗体在病毒粒子表面的结合特异性。通过对整装标本和超薄切片的免疫电子显微镜观察，获得了在痘苗生物发生的各个阶段Ag35与病毒包膜相关联的证据。检测到Ag35作为未成熟和成熟病毒粒子的早期成分，可能存在于双层膜结构中。因此，可以区分Ag35和其他几种痘苗病毒包膜多肽，后者是作为晚期功能合成并在包膜组装后期添加的。
