{"created":"2023-07-27T06:59:59.093170+00:00","id":59752,"links":{},"metadata":{"_buckets":{"deposit":"e505204a-2bbf-4090-a3dc-6ff7e07c3376"},"_deposit":{"created_by":18,"id":"59752","owners":[18],"pid":{"revision_id":0,"type":"depid","value":"59752"},"status":"published"},"_oai":{"id":"oai:kanazawa-u.repo.nii.ac.jp:00059752","sets":["2812:2813:2835"]},"author_link":["32074","20432"],"item_9_biblio_info_8":{"attribute_name":"書誌情報","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"1999-12-07","bibliographicIssueDateType":"Issued"},"bibliographicPageStart":"2p.","bibliographicVolumeNumber":"1997 – 1998","bibliographic_titles":[{"bibliographic_title":"平成10(1998)年度 科学研究費補助金 基盤研究(C) 研究成果報告書概要"},{"bibliographic_title":"1998 Fiscal Year Final Research Report Summary","bibliographic_titleLang":"en"}]}]},"item_9_creator_33":{"attribute_name":"著者別表示","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{}],"nameIdentifiers":[{},{},{}]}]},"item_9_description_21":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"自ら作成したヒト白血球ゲノムDNAコスミドライブラリーをスクリーニングして、Vesicular acetylcholine transporter(VAChT)翻訳領域全長を含むゲノムクローン(Cos25)を得た。VAChT翻訳領域をコードするゲノムにはイントロンがないので、翻訳領域の5′末端と3′末端のポリヌクレオチドをプライマーに用いCos25を鋳型としてpolymerase chain reaction(PCR)を行って、VAChT翻訳領域のみのDNAを得ることができた。このDNAを、インフレームで蛋白発現ベクターpGEX-5X-lに挿入し、スクリーニングした。ところが、得られた大腸菌クローンはすべてVAChT翻訳領域DNAが逆方向に挿入されたプラスミドのみを含んでおり、蛋白誘導可能な大腸菌はクローニングできなかった。そこで、VAChT翻訳領域のカルボキシル基末端側の一部を発現するベクターの作成を試みることに変更した。再度、Cos25を鋳型としてPCRを行い、VAChTカルボキシル基末端側の一部をコードする短いDNAを作成した。これをpGEX-5X-lに挿入して大腸菌をトランスフォームし、ようやくVAChT DNAが正方向にインフレームで組み込まれたクローンを1個(pGEX5Xl-VAChT-3)を得た。このクローンでトランスフォ一ムした大腸菌は、pGEX-5X-l由来の蛋白(グルタチオン-S-トランスフェラーゼ)とVAChTカルボキシル基末端側の58アミノ酸残基との融合蛋白(分子量約36kDa)を産生した。そこで、グルタチオン-S-トランスフェラーゼに対するアフィニティ力ラムを用いて、融合蛋白を発現誘導した大腸菌の細胞破砕液からVAChT蛋白を大量に抽出した。今後、ゲルろ過を行ってヒトVAChT蛋白をさらに精製し、最終的に不純物が混在しないヒトVAChT蛋白を十分量得た後に、ラットに免疫する予定である。","subitem_description_type":"Abstract"},{"subitem_description":"Recently, vesicular acetylcholine transporter (VAChT), the proton-dependent transporter that packages acetylcholine synthesized in the cytoplasm into synaptic vesicles, has been known to be a good marker for morphological identification of cholinergic nerve terminals. In order to investigate how cholinergic nerve terminals are involved in formation of senile plaques in Alzheimer's disease, a specific antibody to human VAChT was tried to be developed by using molecular genetics in this study. High molecular weight DNA was prepared from human blood cells and a genomic library was constructed in pWE cosmid vector after partial digestion of the genomic DNA with Mbo I.A library containing 1.3 X 10^6 independent colonies was screened with the human choline acetyltransferase (ChAT) cDNA obtained in the previous study, because human VAChT gene is located in the first intron of ChAT gene. Thirteen positive clones obtained were rescreened with human VAChT oligonuleotides probes reported, and then a genomic clone containing VAChT gene, Cos 25, was isolated. The full-length DNA of VAChT coding region or the DNA coding the carboxyl terminus was amplified by polymerase chain reaction and subcloned into an expression vector, pGEX-5X- 1. The protein containing glutathione-S-transferase fused with the VAChT carboxyl terminus was successfully expressed in E.coli bacterias. The fusion protein was purified from the bacterial lysates with the affinity matrix Glutathione Sepharose 4B.After further purification of the human VAChT carboxyl terminus with gel filtration for elimination of contaminated bacterial proteins, rats will be immunized for polyclonal antibody production with the human VAGhT carboxyl terminus.","subitem_description_type":"Abstract"}]},"item_9_description_22":{"attribute_name":"内容記述","attribute_value_mlt":[{"subitem_description":"研究課題/領域番号:09670179, 研究期間(年度):1997 – 1998","subitem_description_type":"Other"},{"subitem_description":"出典：研究課題「アルツハイマー型痴呆におけるコリン作動性ニューロンの異常と老人斑との関連 」課題番号09670179\n（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） \n（https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-09670179/096701791998kenkyu_seika_hokoku_gaiyo/）を加工して作成","subitem_description_type":"Other"}]},"item_9_description_5":{"attribute_name":"提供者所属","attribute_value_mlt":[{"subitem_description":"金沢大学医学部","subitem_description_type":"Other"}]},"item_9_identifier_registration":{"attribute_name":"ID登録","attribute_value_mlt":[{"subitem_identifier_reg_text":"10.24517/00066007","subitem_identifier_reg_type":"JaLC"}]},"item_9_relation_28":{"attribute_name":"関連URI","attribute_value_mlt":[{"subitem_relation_name":[{"subitem_relation_name_text":"https://kaken.nii.ac.jp/search/?qm=70169316"}],"subitem_relation_type_id":{"subitem_relation_type_id_text":"https://kaken.nii.ac.jp/search/?qm=70169316","subitem_relation_type_select":"URI"}},{"subitem_relation_name":[{"subitem_relation_name_text":"https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670179/"}],"subitem_relation_type_id":{"subitem_relation_type_id_text":"https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670179/","subitem_relation_type_select":"URI"}},{"subitem_relation_name":[{"subitem_relation_name_text":"https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-09670179/096701791998kenkyu_seika_hokoku_gaiyo/"}],"subitem_relation_type_id":{"subitem_relation_type_id_text":"https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-09670179/096701791998kenkyu_seika_hokoku_gaiyo/","subitem_relation_type_select":"URI"}}]},"item_9_version_type_25":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2022-05-26"}],"displaytype":"detail","filename":"ME-PR-ODA-Y-kaken 1999-2p.pdf","filesize":[{"value":"68.6 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"ME-PR-ODA-Y-kaken 1999-2p.pdf","url":"https://kanazawa-u.repo.nii.ac.jp/record/59752/files/ME-PR-ODA-Y-kaken 1999-2p.pdf"},"version_id":"3409df5d-4432-498f-a1ae-ca8ead3fc2f8"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"research report","resourceuri":"http://purl.org/coar/resource_type/c_18ws"}]},"item_title":"アルツハイマー型痴呆におけるコリン作動性ニューロンの異常と老人斑との関連","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"アルツハイマー型痴呆におけるコリン作動性ニューロンの異常と老人斑との関連"},{"subitem_title":"Cholenirgic nerve terminals and senile plaques in Alzheimer's disease","subitem_title_language":"en"}]},"item_type_id":"9","owner":"18","path":["2835"],"pubdate":{"attribute_name":"公開日","attribute_value":"2022-05-26"},"publish_date":"2022-05-26","publish_status":"0","recid":"59752","relation_version_is_last":true,"title":["アルツハイマー型痴呆におけるコリン作動性ニューロンの異常と老人斑との関連"],"weko_creator_id":"18","weko_shared_id":-1},"updated":"2023-07-27T13:02:20.595414+00:00"}