CAJ | 학술논문

目的：构建人星状病毒非结构蛋白3（ non-structure protein 1a/3， nsP1a/3）基因酵母双杂交诱饵重组质粒，电转化酵母感受态细胞后鉴定酵母转化子特性。方法聚合酶链反应扩增人星状病毒nsP1a/3编码的基因序列，定向插入到穿梭表达载体pGBKT7改造后的pGBST7中，测序确定无误后将其电转化到Y2HGold酵母感受态细胞中，对细胞特性即nsP1a/3-pGBST7诱饵质粒表达产物的毒性和报告基因自激活作用进行鉴定。结果测序结果表明诱饵重组质粒构建正确，阅读框无误。电转化酵母后，其表达产物对酵母细胞无毒性作用，对报告基因亦无激活作用。结论成功构建了用于酵母双杂交的酵母转化子，为研究nsP1a/3蛋白与宿主细胞具体作用的靶蛋白和HAstV在细胞内的复制机制奠定了基础。
목적：구건인성상병독비결구단백3（ non-structure protein 1a/3， nsP1a/3）기인효모쌍잡교유이중조질립，전전화효모감수태세포후감정효모전화자특성。방법취합매련반응확증인성상병독nsP1a/3편마적기인서렬，정향삽입도천사표체재체pGBKT7개조후적pGBST7중，측서학정무오후장기전전화도Y2HGold효모감수태세포중，대세포특성즉nsP1a/3-pGBST7유이질립표체산물적독성화보고기인자격활작용진행감정。결과측서결과표명유이중조질립구건정학，열독광무오。전전화효모후，기표체산물대효모세포무독성작용，대보고기인역무격활작용。결론성공구건료용우효모쌍잡교적효모전화자，위연구nsP1a/3단백여숙주세포구체작용적파단백화HAstV재세포내적복제궤제전정료기출。
Objective To construct a yeast two-hybrid bait vector of non-structure protein 1a/3 (nsP1a/3) of human astrovirus (HAstV), and to characterize the yeast transformants after elec-trotransformation of the recombinant plasmid into the yeast competent cells .Methods The coding sequence of HAstV nsP 1 a/3 obtained by PCR was inserted into the modified plasmid pGST 7.The re-combinant vector nsP1a/3-pGBST7 confirmed by DNA sequencing was transformed into the Y 2 HGo-ld yeast competent cells .The cytotoxicity of the expression products of the recombiant plasmid nsP1a/3-pGBST7 and the autoactivation of the report gene in yeast transformants were exam-ined.Results The sequencing of the nsP 1 a/3-pGBST7 vector showed that the recombinant bait plasmid was successfully constructed and the open reading frame of nsP 1a/3 insert was correct.After electrotransformation, it was found that the expression products of the nsP 1a/3-pGBST7 vector had no cytotoxicity to yeast transformants and also they did not activate the report gene .Conclusion The yeast two-hybrid bait vector of HAstV nsP 1 a/3 was successfully constructed , which laid a foun-dation for further studying the interaction of HAstV nsP 1 a/3 with target proteins and the replication mechanism of HAstV in host cells .