Kobe University · Graduate School of Science and Technology Innovation · 准教授

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• RPN2 gene confers docetaxel resistance in breast cancer2008 · Springer Science and Business Media LLC, 2008年08月, Nature Medicine, 14(9) (9), 939 - 948
• Production of influenza virus-like particles using recombinant insect cellsVirus-like particles (VLPs) are hollow nanoparticles composed of recombinant viral surface proteins without a virus genome. In the present study, we investigated the production of influenza VLPs using recombinant insect cells. DNA fragments encoding influenza A virus hemagglutinin (HA) and matrix protein 1 (M1) were cloned with the Drosophila BiP signal sequence in plasmid vectors containing a blasticidin and a neomycin resistance gene, respectively. After Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with a pair of constructed plasmid vectors, stably transformed cells were established via incubation with blasticidin and G418. Western blot analyses showed that recombinant High Five cells secreted HA and M1 proteins into the culture supernatant. Immunoprecipitation of the culture supernatant with an anti-HA antibody and transmission electron microscopy suggested that secreted HA and M1 proteins were in a particulate structure with a morphology similar to that of an influenza virus. Hemagglutination assay indicated that expressed HA molecules retained hemagglutination activity. In a shake-flask culture, recombinant cells achieved a high HA yield (≈ 10 μg/ml) comparable to the yields obtained using the baculovirus-insect cell system. Recombinant insect cells may serve as excellent platforms for the efficient production of influenza VLPs for use as safe and effective vaccines and diagnostic antigens.
• Production of an antibody Fab fragment using 2A peptide in insect cells.Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells.
• Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction.Recombinant adeno-associated virus (rAAV) is a viral vector commonly used in gene therapy. Residual host cell DNA is an impurity that has been associated with the risk of infection and oncogenicity. Thus, it needs to be monitored for quality control. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) method targeting 18S ribosomal RNA (rRNA) genes to quantitate residual host cell DNA. The copy number of the 18S rRNA gene was determined using two sets of primer pairs for 116- and 247-bp amplicons sharing the C-terminus. For conversion of the copy number of the 18S rRNA gene into the mass concentration of genomic DNA, the accurate copy number of 18S rRNA genes in HEK293 genomic DNA was determined by comparison with copy numbers of three reference genes (EIF5B, DCK, and HBB). Results showed that 88.6-97.9% of HEK293 genomic DNA spiked into rAAV preparations was recovered. The ddPCR-based assay was applied to rAAV preparations to quantitate residual host cell DNA as an impurity. Our findings indicate that the assay can be used for the quantitation and size distribution of residual host cell DNA in rAAV products.

• 胆管細胞癌におけるＢ型肝炎ウイルスゲノム組み込み解析研究 増見 恭子 日本学術振興会, 科学研究費助成事業, 奨励研究, 兵庫医科大学, 2016年04月01日 - 2017年03月31日2016 · KUID parsed section
• 筋疾患予防型トレーニングに遺伝子解析を活用しよう 増見 恭子(小泉恭子), 木下 健司, 村田 成範, 林田 真梨子, 小柳 好生, 中田 研, 米谷 泰一 日本学術振興会, 科学研究費助成事業, 基盤研究(C), 武庫川女子大学, 2012年04月01日 - 2015年03月31日 スポーツ選手にとって靱帯損傷などの故障は人生設計の変更を伴う重大事件であるが、現在までにそれら受傷しやすさを予測するような遺伝子は発見されていない。今回、靱帯損傷などの故障リスクを予測し予防することを目的として、本学運動部99名を対象にスポーツや筋肉に関わる遺伝子型検査およびアンケート調査を実施した。その結果ACTN3遺伝子の577Rは577Xよりも有意に筋疾患歴を持つことが判明した。この結果より、ACTN3遺伝子多型を調べることで自身の受傷リスクが分かる可能性が示唆された。今後この結果をもとに、筋疾患を回避するためのトレーニング方法を検討していく予定である。2012 · KUID parsed section
• 次世代への遺伝子および遺伝子検査教育のための研究 村田 成範, 木下 健司, 増見 恭子, 林田 真梨子 日本学術振興会, 科学研究費助成事業, 基盤研究(C), 武庫川女子大学, 2011年 - 2013年 本研究における教育活動として、当初の高等学校での出前講義・実験および大学での初年次教育に加え、小学生とその保護者、飲料関連企業、薬局などで、遺伝子検査を含むゲノム科学リテラシーセミナーを実施し、遺伝子に関する教育方法および解析技術を改良した。アルコール遺伝子検査を行った大学では３年次以降での追跡調査も開始した。教育方法の改善に加えて、高校での遺伝子実験法の改良により、生徒自身が実験を主導できる実験系プロトコールを開発し、個人情報取り扱いを含めた高校生主体プロジェクトの開始に向けた検討を行った。また大学でのアドバンス科目用として遺伝子情報取り扱いのための基礎技術も開発した。2011 · KUID parsed section

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