Boulter N, Knight P A, Hunt P D, Hennessey E S, Katzer F, Tait A, Williamson S, Brown D, Baylis H A, Hall R
Department of Biology, University of York, UK.
Parasite Immunol. 1994 Feb;16(2):97-104. doi: 10.1111/j.1365-3024.1994.tb00328.x.
SPAG-1 is a surface antigen on Theileria annulata sporozoites that is a candidate both for inclusion in a subunit vaccine and as a ligand for host cell recognition. We have pinpointed major neutralizing epitopes to the C terminus. To facilitate this we expressed SPAG-1 as a series of defined fragments in the pGEX system. These constructs were validated by sequencing and by their spectrum of reactivity with monoclonal antibody (MoAb) BA4. This MoAb recognizes the elastin motif VGVAPG, that is predicted to occur three times in the N terminal half of SPAG-1. The recombinant proteins were then tested by Western blotting with a neutralizing MoAb (1A7) and two neutralizing bovine sera (10T and 34A). The results demonstrate that 1A7 and the bovine sera react with determinants unique to the C terminus. We mapped the neutralizing determinant recognized by MoAb 1A7 to a 16 residue sequence (residues 807-822) using synthetic peptides. Interestingly the bovine sera do not recognize the 1A7 epitope. The potential role of the C terminus as a ligand for host cell recognition and the implications for sub-unit vaccine production are discussed.
SPAG-1是环形泰勒虫子孢子表面的一种抗原，它既是亚单位疫苗的候选成分，也是宿主细胞识别的配体。我们已确定主要的中和表位位于C末端。为便于研究，我们在pGEX系统中将SPAG-1表达为一系列特定片段。这些构建体通过测序以及它们与单克隆抗体（MoAb）BA4的反应谱进行了验证。该单克隆抗体识别弹性蛋白基序VGVAPG，预计在SPAG-1的N末端一半区域出现三次。然后用一种中和性单克隆抗体（1A7）和两种中和性牛血清（10T和34A）通过蛋白质印迹法对重组蛋白进行检测。结果表明，1A7和牛血清与C末端特有的决定簇发生反应。我们使用合成肽将单克隆抗体1A7识别的中和决定簇定位到一个16个残基的序列（残基807 - 822）。有趣的是，牛血清不识别1A7表位。讨论了C末端作为宿主细胞识别配体的潜在作用以及对亚单位疫苗生产的影响。
