文昌鱼BRA蛋白的原核表达及多克隆抗体制备

Prokaryotic Expression of Amphioxus BRA Protein and Preparation of the Polyclonal Antibody

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p ET28a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Ni2+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50μg/(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 Brachyuryhomologues 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 p ET28a（Fig. 2b）. The recombinant plasmid p ET28a-Bra2-N was transformed intoEscherichia 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 Ni2＋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.