Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil.
Vet Parasitol. 2012 Jul 6;187(3-4):521-8. doi: 10.1016/j.vetpar.2012.01.027. Epub 2012 Jan 28.
Rhipicephalus (Boophilus) microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. The large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5mg/L of culture, respectively. Purified boophilin inhibited trypsin (K(i) 0.65 nM), neutrophil elastase (K(i) 21 nM) and bovine thrombin (K(i) 57 pM), while D1 inhibited trypsin and neutrophil elastase (K(i) of 2.0 and 129 nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control.
微小牛蜱是一种外寄生虫，会导致肉类、牛奶和皮革产量下降，这既是由于牛失血，也是由于传播无形体病和巴贝斯虫病引起的。微小牛蜱是丝氨酸蛋白酶抑制剂的丰富来源，包括胰蛋白酶抑制剂 BmTI-A 和 BmTI-6、枯草杆菌蛋白酶抑制剂 BmSI，以及最近描述的凝血酶抑制剂，boophilin。Boophilin 是一种双 Kunitz 型凝血酶抑制剂，具有与第二种（非凝血酶）丝氨酸蛋白酶分子形成三元复合物的不寻常能力。报告了在巴斯德毕赤酵母中大规模表达和纯化 boophilin 及其分离的 N 端（D1）结构域、表达谱以及 RNAi 介导的基因沉默对蜱卵生产的影响。全长 boophilin 和 D1 的表达量分别为 21 和 37.5mg/L 的培养物。纯化的 boophilin 抑制胰蛋白酶（K(i)0.65 nM）、中性粒细胞弹性蛋白酶（K(i)21 nM）和牛凝血酶（K(i)57 pM），而 D1 抑制胰蛋白酶和中性粒细胞弹性蛋白酶（K(i)分别为 2.0 和 129 nM），但不抑制凝血酶。使用 RNAi 沉默 boophilin 基因导致卵重产量减少 20%，表明在该生命阶段表达 boophilin 虽然很重要，但并非必不可少，这可能是由于与微小牛蜱中肠中的其他丝氨酸蛋白酶抑制剂存在功能重叠。考虑到我们的数据，Boophilin 可能与其他抗原结合用于生产控制蜱的疫苗。
