イヌジステンパーウイルスの病原性解析と遺伝子治療への応用

犬瘟热病毒病原分析及其在基因治疗中的应用

• 批准号: 19J14751
• 负责人: 南 昌平
• 金额: $ 1.09万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2019
• 资助国家: 日本
• 起止时间: 2019-04-25 至 2021-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-19J14751/
• 关键词: イヌジステンパーウイルス; リバースジェネティクス; ウイルスベクター; 感染性クローン

昨年に確立したイヌジステンパーウイルス（CDV）のリバースジェネティクス系を応用させ、ウイルスベクターとして細胞への遺伝子導入を試みた。始めに、CDVのウイルスゲノム内全ORFの欠損体作製を試みた。T7プロモーター、ハンマーヘッド型リボザイム、D型肝炎ウイルスリボザイムおよびT7ターミネーターを含むプラスミドベクターにCDVの5'UTRと3'UTR間にEGFP発現遺伝子をクローニングした。得られたプラスミドをCAGプロモーターによる全CDV蛋白発現プラスミドと共にBHK/T7-9細胞へトランスフェクションし、培養上清を回収した。その後、培養上清をA72/cSLAM細胞へと接種し、EGFP蛋白の蛍光を観察した。結果、EGFPのシグナルは得られなかった。パラミクソウイルス科は各ORFの上流UTRにあるgene start(GS)、下流UTRにあるgene end(GE)と呼ばれるシグナル配列を認識してウイルスポリメラーゼが転写を開始、終止することが知られている。本実験ではGSはCDV N遺伝子のものであり、GEはCDV L遺伝子のものであったため、転写が行われなかったと考えられた。そこで、GEの配列をN遺伝子のものに置換したプラスミドを作製し、同様の実験を行ったが、EGFPのシグナルは得られなかった。GS、GEの配列の組み合わせやCDVのパッケージングシグナル配列の欠如が原因と考えられた。次にH遺伝子欠損CDVの作製を試みた。H遺伝子をEGFP遺伝子に置換したCDVゲノムを感染性粒子作製と同様の手法に加え、H蛋白を過剰発現させ実施したが、EGFPのシグナルは得られなかった。しかしながら、上清を用いたウエスタンブロットによる抗原検出ではF蛋白が検出されたため、EGFP遺伝子が発現していないことが考えられた。上述のGS、GEやコドンの最適化などが改善点として挙げられる。

Last year, we made sure that we were able to make sure that we were able to use the computer to make sure that we were in an attempt. Last year, we made sure that we did not know if we were going to make use of the computer. Last year, we made sure that we were able to make sure that we were using the CDV to make sure that we were using the computer. Since the beginning of the year, the CDV will have to make an attempt to ensure that all ORF employees are in arrears. T7 virus infection, hepatitis D virus infection. The whole CDV protein was detected in the whole CAG protein. The total BHK/T7-9 cells were detected. The supernatant was incubated. After incubation, the supernatant of A72/cSLAM cells was inoculated and EGFP protein was examined by light microscopy. Results: the results showed that there were significant differences between the two groups of EGFP patients. For all ORF users, the upstream UTR transmission gene start (GS) and the downstream UTR communication gene end (GE) are required to read the information and start the writing process, so that the information is known. This article is about GS CDV N, GE CDV L, CDV L, and writing lines. The following lines are assigned: n, GE, and so on. Please do the same thing as you would like to do, and you will need to know that you will not be able to do so if you want to do so. The configuration of GS and GE is not as good as the reason for the failure of the arrangement of GS and GE. For the second time, you are not allowed to CDV to make an attempt. H-EGFP was used to treat the infectious particles of CDV virus, H-protein was used to treat infectious particles, H-protein was used to treat infectious particles, H-protein was used to treat infectious particles, H-protein was used to treat infectious particles, H-protein was used to treat infectious particles, H-protein was used to treat infectious particles, and H-protein was used to treat infectious particles. In the supernatant, the antigens were detected in the supernatant, and the F protein was detected in the supernatant, while the F protein was detected in the supernatant. the supernatant and the supernatant were detected in the supernatant, the supernatant and the supernatant. The above GS, GE optimization, improvement point, improvement point.

项目成果

Kasetsart University(タイ)

农业大学（泰国）

Histopathological Characterization of Cases of Spontaneous Fatal Feline Severe Fever with Thrombocytopenia Syndrome, Japan.

• DOI: 10.3201/eid2704.204148
• 发表时间: 2021-04
• 期刊: Emerging infectious diseases
• 影响因子: 11.8
• 作者: Sakai Y;Kuwabara Y;Ishijima K;Kagimoto S;Mura S;Tatemoto K;Kuwata R;Yonemitsu K;Minami S;Kuroda Y;Baba K;Okuda M;Shimoda H;Sakurai M;Morimoto M;Maeda K
• 通讯作者: Maeda K