Ad-ERα-36-Fc-GFP的制备及鉴定  

Preparation and characterization of Ad-ERα-36-Fc-GFP

作　　者：谢雨琼 李春春[1] 李晓烨 陈丽红[1] 严毛晓[2] 曹江[1] XIE Yuqiong;LI Chunchun;LI Xiaoye;CHEN Lihong;YAN Maoxiao;CAO Jiang(Clinical Research Center,the Second Affiliated Hospital,School of Medicine,Zhejiang University,Hangzhou 310009,Zhejiang,China;Department of Otorhinolaryngology,the Second Affiliated Hospital,School of Medicine,Zhejiang University,Hangzhou 310009,Zhejiang,China)

机构地区：[1]浙江大学医学院附属第二医院临床研究中心,浙江杭州310009 [2]浙江大学医学院附属第二医院耳鼻咽喉科,浙江杭州310009

出　　处：《生物工程学报》2022年第3期1086-1095,共10页Chinese Journal of Biotechnology

基　　金：浙江省自然科学基金(LQ19H160017,LY18H160006)。

摘　　要：ERα-36是雌激素受体α新亚型,促进肿瘤细胞增殖、侵袭及耐药,可作为治疗靶点,但目前仅有小分子靶向药物研发。利用重组腺病毒携带诱饵受体,可进行靶向ERα-36的基因治疗探索。首先通过基因工程技术,构建可表达由Igκ信号肽引导分泌的重组融合蛋白ERα-36-Fc的穿梭质粒pDC316-Igκ-ERα-36-Fc-GFP;利用AdMax;腺病毒包装系统进行Ad-ERα-36-Fc-GFP腺病毒的包装、鉴定及扩增;感染三阴性乳腺癌细胞MDA-MB-231并进行表达及功能验证。结果表明成功制备Ad-ERα-36-Fc-GFP重组腺病毒,可高效感染三阴性乳腺癌细胞MDA-MB-231,表达并分泌ERα-36-Fc重组蛋白,显著抑制EGFR/ERK信号通路。Ad-ERα-36-Fc-GFP腺病毒的制备为进一步开展靶向ERα-36的肿瘤基因治疗研究奠定基础。ERα-36 is a novel subtype of estrogen receptorαwhich promotes tumor cell proliferation,invasion and drug resistance,and it serves as a therapeutic target.However,only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present.Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor.The recombinant shuttle plasmid pDC316-Igκ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Igκ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc.The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged,characterized and amplified using AdMax;adenovirus packaging system.The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells.Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated.The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc,leading to significant inhibition of EGFR/ERK signaling pathway.Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.

关 键 词：Ad-ERα-36-Fc-GFP 腺病毒制备 诱饵受体 基因治疗 

分 类 号：R730.5[医药卫生—肿瘤] Q78[医药卫生—临床医学]