Cardoso Thyago H S, Lu Stephen, Gonzalez Boris R G, Torquato Ricardo J S, Tanaka Aparecida S
Department of Biochemistry, Escola Paulista de Medicina, Universidade de Federal de São Paulo (UNIFESP), Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil.
Department of Biochemistry, Escola Paulista de Medicina, Universidade de Federal de São Paulo (UNIFESP), Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil.
Biochimie. 2017 Sep;140:117-121. doi: 10.1016/j.biochi.2017.07.005. Epub 2017 Jul 21.
The Rhipicephalus (Boophilus) microplus is an exclusive bovine ectoparasite responsible for the transmission of pathogens that decrease meat, leather and milk productions. Cattle vaccination is an alternative to control tick infestations, but the discovery of potential antigens is still a challenge for researchers. Recently, our group performed a midgut transcriptome of engorged R. microplus tick, and out of 800 ESTs sequences one cystatin-coding sequence was identified and named Rmcystatin-4. In order to understand the physiological role of Rmcystatin-4, the aim of this work was the expression, purification and functional characterization of a novel type 2 cystatin from the tick R. microplus. Rmcystatin-4 gene expression was identified mostly in tick midgut suggesting its possible role in blood digestion control. Our data showed that rRmcystatin-4 was successfully expressed in active form using Pichia pastoris system and the purified inhibitor presented high selectivity to BmCl-1 (Ki = 0.046 nM). Moreover, rRmcystatin-4 was able to impaired BmCl-1 activity towards bovine hemoglobin.
微小扇头蜱（牛蜱属）是一种专门寄生在牛身上的体外寄生虫，可传播导致肉类、皮革和牛奶产量下降的病原体。牛疫苗接种是控制蜱虫侵扰的一种替代方法，但发现潜在抗原对研究人员来说仍是一项挑战。最近，我们小组对饱血的微小扇头蜱进行了中肠转录组分析，在800个EST序列中鉴定出一个胱抑素编码序列，并将其命名为Rmcystatin-4。为了了解Rmcystatin-4的生理作用，本研究的目的是对微小扇头蜱的一种新型2型胱抑素进行表达、纯化和功能表征。Rmcystatin-4基因表达主要在蜱的中肠中被鉴定出来，这表明它可能在血液消化控制中发挥作用。我们的数据表明，重组Rmcystatin-4（rRmcystatin-4）利用毕赤酵母系统成功表达为活性形式，纯化后的抑制剂对BmCl-1具有高选择性（Ki = 0.046 nM）。此外，rRmcystatin-4能够抑制BmCl-1对牛血红蛋白的活性。
