Brachyury编码的转录调控因子BRA参与脊索动物脊索的分化形成,文昌鱼是最早具有真正脊索的后生动物类群,因此开展文昌鱼Brachyury基; 因功能研究,将有助于揭示脊索的起源与进化。文昌鱼具有2个Brachyury基因:Bra1和Bra2,二者编码的蛋白序列相似度高达93%,缺少有效; 区分二者的特异抗原表位,转录组数据分析表明Bra2表达量显著高于Bra1,进一步对BRA2蛋白序列特征分析发现其N端拥有丰富的潜在抗原决定簇,因; 此本研究选择了Bra2 N端696; bp基因序列所编码的蛋白片段作为制备抗体的抗原蛋白。将该段基因序列克隆重组入pET28a原核表达质粒,经诱导表达获分子量约31; ku的可溶性重组蛋白。通过Ni~(2+)亲和层析柱纯化,得到1.3 g/L高纯度抗原蛋白,用3只ICR小鼠(Mus; musculus)经4轮重组蛋白免疫[剂量50 mug/(只·次)]后获得最高效价(1︰256; 000)的多克隆抗体。Western印迹结果显示,本研究制备的鼠抗文昌鱼BRA多克隆抗体不仅可特异识别重组抗原蛋白,也可高效识别文昌鱼胚胎总蛋白; 中的BRA1和BRA2,为后续深入研究文昌鱼BRA在脊索发育调控中的作用提供了有力的分子工具。Homologues of the T-box gene Brachyury play an essential role in; notochord specification of early embryo development of chordates.; Amphioxuses, also called cephalochordates, represent the most basally; divergent lineage of chordates, being the sister group of urochordates; and vertebrates. It is now generally agreed that amphioxus is the first; animal group to evolve a real sense of notochord in all metazoans, hence; studying Brachyury in amphioxus would shed important insights into the; origin and evolution of notochord. In order to explore the function of; Brachyury in the developmental regulation network of amphioxus, we; generated a mouse anti-amphioxus BRA polyclonal antibody. Amphioxus; possesses two Brachyury homologues genes: Bra1 and Bra2, encoding two; proteins with a sequence similarity of 93% (Fig. 1a). It is very hard to; distinguish specific epitopes between them. The transcriptome data; showed that the expression level of Bra2 was much higher than that of; Bra1 (Fig. 1b). Further analysis of BRA2 sequence feature indicated that; ideal presume antigenic determinants located in the N-terminal of BRA2; (Fig. 1c). Therefore, a 696 bp gene segment was amplified by PCR from; Bra2 N-terminal (Fig. 2a) and inserted into prokaryotic expression; vector pET28a (Fig. 2b). The recombinant plasmid pET28a-Bra2-N was; transformed into Escherichia coli BL21 and induced to express the tagged; protein (Fig. 3a). We obtained a huge amount of soluble recombinant; protein with an expected size (31 ku) (Fig. 3b) and purified the tagged; protein using Ni~(2+) affinity chromatography (Fig. 3c). Three ICR mice; were immunized to generate polyclonal antibodies against amphioxus BRA2; with purified recombinant protein (1.3 g/L). The enzyme-linked; immunosorbent assay (ELISA) showed that the titer of mouse; anti-amphioxus BRA2 antibody was 1︰256 000, with a high sensitivity; (Fig. 4a). Western blot experiments showed that the polyclonal antibody; could not only effectively identify recombinant protein (Fig. 4b) but; also recognized amphioxus BRA1 and BRA2 (Fig. 4c, Fig. 5a, b). In; conclusion, we successfully generated the mouse anti-amphioxus BRA; polyclonal antibody that would be a powerful molecular tool for further; investigating the function of Brachyury in amphioxus.国家自然科学基金项
