Tick Vaccine Prevention of Lyme Borreliosis

描述（由申请人提供）：我们的策略是通过干扰蜱虫进食的能力来防止莱姆疏螺旋体病的传播。拟议的项目试图识别和描述硬蜱蜱使用的抗凝血剂家族，以允许其以哺乳动物宿主为食。这种抗凝剂对于蜱的成功进食和蜱传病原体的传播至关重要。我们将通过从蜱唾液腺和唾液中生化分离，并通过使用适当的引物片段探测 cDNA 和基因组文库来探索蜱抗凝剂家族。我们实验室之前曾尝试通过使用来自蜱免疫动物的抗体探测 cDNA 文库来分离蜱唾液蛋白，结果产生了 40 个克隆；其中 22 种以我们称为 SALP-14 的抗凝剂为代表。在本提案中，我们将使用源自先前鉴定的克隆的同源部分的引物来探测文库，以表征我们现在认为对蜱进食和莱姆疏螺旋体病传播至关重要的抗凝剂家族。我们将表达和纯化重组抗凝剂并表征其活性。为了确定活性位点，我们将生成这些抗凝剂的缺失突变体，并确定有效催化活性和抗原性所必需的最小区域。目标是通过针对抗凝血分子功能表位的免疫反应来干扰进食。吃饱对于蜱虫完成其生命周期以及传播其携带的病原体至关重要。

DESCRIPTION (provided by applicant): Our strategy is to prevent transmission of Lyme borreliosis by interfering with the tick's ability to feed to repletion. The proposed project attempts to identify and characterize a family of anticoagulants used by Ixodes ticks needed to permit feeding on their mammalian hosts. Such anticoagulants are vital to the success of tick feeding and in the transmission on tick borne pathogens. We shall explore the family of Ixodes tick anticoagulants by biochemical separation from tick salivary gland and saliva, and by probing cDNA and genomic libraries with appropriate primer fragments. Previous attempts in our laboratories to isolate tick salivary proteins by probing cDNA libraries with antibodies from animals made tick immune resulted in 40 clones; 22 of which were represented by an anticoagulant that we have called SALP-14. In the present proposal we will probe libraries with primers derived from homologous portions of the clones previously identified in order to characterize what we now recognize as a family of anticoagulants vital to tick feeding and transmission of Lyme borreliosis. We will express and purify the recombinant anticoagulants and characterize their activities. To determine active sites, we will generate deletion mutants of these anticoagulants and identify the smallest region essential for effective catalytic activity, as well as antigenicity. The goal is to interfere with feeding by the immune response directed against the functional epitope(s) of the anticoagulant molecules. Feeding to repletion is essential for ticks to complete their life cycle and for the transmission of the pathogens they harbor.