东方巴贝斯虫硫氧还蛋白过氧化物酶 2 基因克隆及表达

摘要

硫氧还蛋白过氧化物酶广泛存在于原核及真核生物中,是生物抗氧化系统的重要成员。该类酶通过半胱氨酸依赖的催化机制,在抵御氧化应激过程中发挥关键作用。在东方巴贝斯虫( Babesia orientalis)的 cDNA 及 gDNA 中克隆到硫氧还蛋白过氧化物酶 2(BoTPx-2)基因。该基因开放阅读框(ORF)长度为 771 bp,无内含子,编码 257 个氨基酸,预测蛋白分子量约为 27.7 ku,等电点为 8.86。其编码的氨基酸序列包含两个保守的半胱氨酸结构域 Phe-Val-Cys-Pro 和 Glu-Val-Cys-Pro,分别位于第 107 位和第 227 位。生物信息学分析表明,该蛋白具有高度保守的催化结构域,属于 PTZ00137 超家族。通过将 BoTPx-2 与其他巴贝斯虫、泰勒虫及疟原虫的 TPx-2 序列进行比对及系统进化树分析,发现其与牛巴贝斯虫 TPx-2(XM_001609049)的亲缘关系最近,序列相似度达 79.44%。利用同源重组方法构建重组表达质粒 BoTPx-2-pE-sumo,该质粒包含 1 个 His 标签,编码 364 个氨基酸,分子量约为 40 ku。经 IPTG 诱导后,重组蛋白以可溶性形式大量表达于上清。进一步的蛋白质免疫印迹(Western blot)结果显示,感染东方巴贝斯虫水牛阳性血清可特异性识别大小约 40 ku 的条带,与重组蛋白大小一致,表明 BoTPx-2 具有良好的免疫原性,可引起感染水牛产生相应的免疫反应。此外,利用 BoTPx-2-pE-sumo 重组蛋白制备的多克隆抗体,在东方巴贝斯虫全虫抗原中检测到一条约 27 ku 的特异性条带,与 BoTPx-2 的预期分子量相符,即为 BoTPx-2 的天然大小。研究结果成功鉴定并初步分析了东方巴贝斯虫 TPx-2 基因及其表达产物,为后续深入研究该虫的抗氧化机制提供理论基础。

关键词

硫氧还蛋白过氧化物酶 / 东方巴贝斯虫 / 半胱氨酸结构域 / 免疫原性

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基金资助

黑龙江省自然科学基金联合引导项目(LH2024C019)

黑龙江省博士后面上项目(LBH-Z22084)

东农学者计划“青年才俊”项目(54970312)