Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia 4072, Queensland, Australia.
J Virol Methods. 2011 Jul;175(1):20-7. doi: 10.1016/j.jviromet.2011.04.010. Epub 2011 Apr 27.
Previous studies have concluded that the Flavivirus prM protein is a suitable viral antigen to distinguish serologically between infections with closely related Flaviviruses (Cardosa et al., 2002). To express the recombinant West Nile virus (WNV) prM antigen fused to a suitable affinity tag for purification, a series of prM-His-tag and prM-V5-tag fusion proteins were generated. Analysis of the prM-His-tag fusion proteins revealed that either prM epitopes were disrupted or the His-tag was not presented properly depending on the location of the His tag and the presence of the prM transmembrane domains in these constructs. This identified domains critical for proper folding of prM, and arrangements that allowed the correct presentation of the His-tag. However, the inclusion of the V5 epitope tag fused to the C terminus of prM allowed formation of the authentic antigenic structure of prM and the proper presentation of the V5 epitope. Capture of tagged recombinant WNV(NY99) prM antigen to the solid phase with anti-V5 antibody in ELISA enabled the detection of prM-specific antibodies in WNV(NY99)-immune horse serum, confirming its potential as a useful diagnostic reagent.
先前的研究表明，黄病毒前膜蛋白（prM）是一种合适的病毒抗原，可用于区分血清学上密切相关的黄病毒感染（Cardosa 等人，2002 年）。为了表达与合适的亲和标签融合的重组西尼罗河病毒（WNV）prM 抗原，以用于纯化，生成了一系列 prM-His 标签和 prM-V5 标签融合蛋白。对 prM-His 标签融合蛋白的分析表明，无论是 prM 表位被破坏，还是 His 标签未正确呈现，这取决于 His 标签的位置和这些构建体中 prM 跨膜结构域的存在。这确定了 prM 正确折叠所必需的结构域，以及允许正确呈现 His 标签的排列。然而，将 V5 表位标签融合到 prM 的 C 末端，允许形成 prM 的真实抗原结构，并正确呈现 V5 表位。通过在 ELISA 中用抗 V5 抗体将标记的重组 WNV（NY99）prM 抗原捕获到固相上，可检测到 WNV（NY99）免疫马血清中的 prM 特异性抗体，证实其作为有用诊断试剂的潜力。
