Cooperative Research Centre for Beef Genetic Technologies, Armidale, NSW, Australia.
Ticks Tick Borne Dis. 2012 Jun;3(3):159-69. doi: 10.1016/j.ttbdis.2012.03.002. Epub 2012 May 17.
Rhipicephalus microplus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZαA vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1). qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle.
微小牛蜱是一种重要的牛体外寄生虫，广泛分布于世界的热带和亚热带地区，给养牛业造成了巨大的经济损失。它作为一种外寄生虫的成功与其能够解除宿主抗止血和抗炎反应的能力有关。丝氨酸蛋白酶抑制剂（serpins）在宿主-寄生虫相互作用的调节中具有重要作用。通过 RACE 分离并随后克隆到 pPICZαA 载体中的微小牛蜱丝氨酸蛋白酶抑制剂（R. microplus serpins）cDNA。对 cDNA 和预测的氨基酸序列分析表明，该 cDNA 具有保守的丝氨酸蛋白酶抑制剂结构域。使用生物信息学工具预测了 B 细胞和 T 细胞表位。甲醇诱导 0.2mg/L 后，重组微小牛蜱丝氨酸蛋白酶抑制剂（rRMS-3）被分泌到毕赤酵母的培养物中。组织和生命周期阶段的 qRT-PCR 表达分析表明，RMS-3 主要在雌性成蜱的唾液腺中表达。使用抗蜱和易感牛血清对 rRMS-3 和预测的 B 细胞表位进行免疫识别。只有抗蜱牛血清识别 B 细胞表位 AHYNPPPPIEFT（Seq7）。RMS-3 在毕赤酵母中表达，ELISA 筛选也显示出对抗蜱牛血清的更高识别。结果表明，RMS-3 高度特异性地分泌到抗蜱牛吸食的微小牛蜱的叮咬部位。用抗 AHYNPPPPIEFT 绵羊血清对半饱吸血的微小牛蜱进行毛细管喂养，导致微小牛蜱的繁殖能力降低 81.16%。因此，可以得出结论，微小牛蜱丝氨酸蛋白酶抑制剂（RMS-3）在宿主-寄生虫相互作用中具有重要作用，可克服抗性牛的免疫反应。
