Nuclear filament-containing protein 2 (Nuf2p), a centromere-associated protein, is an indispensable component of Ndc80 complex, which is important for stable microtubule–kinetochore attachment, chromosome pairing, and spindle assembly in mitosis. Based upon contig sequences coding for Nuf2p from a pyrosequencing transcriptome of Saccharina japonica, primers were designed for molecular cloning of this kinetochore protein. The open reading frame (ORF) of this gene SjNuf2 was 1,452 bp long, encoding a putative protein consisting of 483 amino acids. Multi-sequence alignment showed SjNuf2p possessed several conserved domains of Nuf2, Spc7, SPT2, and MIT. To illustrate the location of SjNuf2p using immunocytochemistry, its ORF was inserted into a prokaryotic expression vector pET-28a. Then, the recombinant SjNuf2p (rSjNuf2p) was induced to be expressed in Escherichia coli. The rSjNuf2p was verified by Western blotting with the commercial anti-6 × His-tag antibody and then purified using affinity chromatography. The polyclonal antibody of anti-SjNuf2p was raised against the purified rSjNuf2p. Western blotting with this prepared antibody showed the presence of SjNuf2p in the gametophytes of a Saccharina cultivar. SjNuf2p and α/β-tubulin were subsequently co-localized to the paraffin sections of kelp chromosomes with their corresponding antibodies, thus illustrating that SjNuf2p could reside at the kelp centromeres. This study laid a solid foundation for kelp centromere and karyotype analyses.

中文翻译：

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糖精 (Phaeophyta) 品种配子体染色体上动粒蛋白 Nuf2p 的免疫细胞化学定位

含核丝蛋白 2 (Nuf2p) 是一种着丝粒相关蛋白，是 Ndc80 复合物不可或缺的组成部分，对于稳定的微管-动粒附着、染色体配对和有丝分裂中的纺锤体组装很重要。基于编码来自日本糖精焦磷酸测序转录组的 Nuf2p 的重叠群序列，设计了用于该动粒蛋白的分子克隆的引物。该基因 SjNuf2 的开放阅读框 (ORF) 长 1,452 bp，编码由 483 个氨基酸组成的推定蛋白质。多序列比对显示 SjNuf2p 拥有 Nuf2、Spc7、SPT2 和 MIT 的几个保守域。为了使用免疫细胞化学来说明 SjNuf2p 的位置，将其 ORF 插入到原核表达载体 pET-28a 中。然后，重组 SjNuf2p (rSjNuf2p) 在大肠杆菌中被诱导表达。rSjNuf2p 用商业抗 6 × His 标签抗体通过蛋白质印迹验证，然后使用亲和层析纯化。抗-SjNuf2p 的多克隆抗体是针对纯化的 rSjNuf2p 产生的。用这种制备的抗体进行的蛋白质印迹表明，糖精栽培品种的配子体中存在 SjNuf2p。SjNuf2p 和 α/β-微管蛋白随后与其相应的抗体共定位到海带染色体的石蜡切片上，从而说明 SjNuf2p 可以位于海带着丝粒处。该研究为海带着丝粒和核型分析奠定了坚实的基础。rSjNuf2p 用商业抗 6 × His 标签抗体通过蛋白质印迹验证，然后使用亲和层析纯化。抗-SjNuf2p 的多克隆抗体是针对纯化的 rSjNuf2p 产生的。用这种制备的抗体进行的蛋白质印迹表明，糖精栽培品种的配子体中存在 SjNuf2p。SjNuf2p 和 α/β-微管蛋白随后与其相应的抗体共定位到海带染色体的石蜡切片上，从而说明 SjNuf2p 可以位于海带着丝粒处。该研究为海带着丝粒和核型分析奠定了坚实的基础。rSjNuf2p 用商业抗 6 × His 标签抗体通过蛋白质印迹验证，然后使用亲和层析纯化。抗-SjNuf2p 的多克隆抗体是针对纯化的 rSjNuf2p 产生的。用这种制备的抗体进行的蛋白质印迹表明，糖精栽培品种的配子体中存在 SjNuf2p。SjNuf2p 和 α/β-微管蛋白随后与其相应的抗体共定位到海带染色体的石蜡切片上，从而说明 SjNuf2p 可以位于海带着丝粒处。该研究为海带着丝粒和核型分析奠定了坚实的基础。SjNuf2p 和 α/β-微管蛋白随后与其相应的抗体共定位到海带染色体的石蜡切片上，从而说明 SjNuf2p 可以位于海带着丝粒处。该研究为海带着丝粒和核型分析奠定了坚实的基础。SjNuf2p 和 α/β-微管蛋白随后与其相应的抗体共定位到海带染色体的石蜡切片上，从而说明 SjNuf2p 可以位于海带着丝粒处。该研究为海带着丝粒和核型分析奠定了坚实的基础。