Ad-ERα-36-Fc-GFP的制备及鉴定

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

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-Ig κ-ERα-36-Fc-GFP；利用AdMax^TM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。

Abstract:

ERα-36 is a novel subtype of estrogen receptor α which 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-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-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^TM 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.