College of Veterinary Medicine, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Hunan Collaborative Innovation Center for Safety Production of Livestock and Poultry, Hunan Agricultural University, Changsha, China.
Med Vet Entomol. 2021 Mar;35(1):42-50. doi: 10.1111/mve.12467. Epub 2020 Aug 4.
Hemalin, initially described in Haemaphysalis longicornis, is a protein with anticoagulant activity. We retrieved a gene fragment functionally annotated as hemalin from H. flava salivary gland transcriptomic library, but its full-length complementary DNA (cDNA) and antithrombogenicity have not been investigated in the species. Here we cloned the full length of hemalin (Hf-hemalin) by 3'-end rapid-amplification of cDNA ends, and the open reading frame (ORF) of Hf-hemalin was expressed in Escherichia coli. The recombinant protein (rHf-Hemalin) was tested for antithrombogenicity. The full-length of Hf-hemalin was 607 bp with an ORF of423 bp. Protein encoded by Hf-hemalin was predicted to contain 2 Kunitz domains and a signal peptide. The expression of Hf-hemalin in salivary glands, midguts and ovaries was higher in the semi-engorged than the fully engorged. Prokaryotic expression yielded a product of 40 kDa containing a glutathione S-transferase (GST) tag. Incubation of rHf-Hemalin with rat plasma significantly extended prothrombin time and activated partial thromboplastin time compared with normal saline and GST controls. Our data demonstrated that Hemalin from H. flava shared a similar primary structure with that from H. longicornis, and was also anticoagulant. Further investigations are needed to test its feasibility to be an antigen candidate for the development of vaccines against ticks.
血纤蛋白原，最初在长角血蜱中被描述，是一种具有抗凝活性的蛋白质。我们从黄足硬蜱唾液腺转录组文库中检索到一个功能注释为血纤蛋白原的基因片段，但该基因在该物种中的全长 cDNA 和抗血栓形成特性尚未被研究。在这里，我们通过 3' 端快速扩增 cDNA 末端克隆了血纤蛋白原（Hf-hemalin）的全长，并在大肠杆菌中表达了 Hf-hemalin 的开放阅读框（ORF）。重组蛋白（rHf-Hemalin）进行了抗血栓形成活性检测。Hf-hemalin 的全长为 607bp，ORF 为 423bp。Hf-hemalin 编码的蛋白质被预测含有 2 个 Kunitz 结构域和一个信号肽。半饱血期黄足硬蜱唾液腺、中肠和卵巢中的 Hf-hemalin 表达高于饱血期。原核表达产生了一个 40kDa 的产物，含有谷胱甘肽 S-转移酶（GST）标签。与生理盐水和 GST 对照相比，rHf-Hemalin 与大鼠血浆孵育显著延长了凝血酶原时间和部分促凝血酶原时间。我们的数据表明，黄足硬蜱的血纤蛋白原与长角血蜱的血纤蛋白原具有相似的一级结构，并且也具有抗凝活性。需要进一步的研究来测试其作为抗蜱疫苗抗原候选物的可行性。
