靶向性抗肿瘤融合基因RGD-IL-24重组腺病毒的构建及鉴定

中国临床药理学与治疗学 ›› 2010, Vol. 15 ›› Issue (11): 1223-1228.

靶向性抗肿瘤融合基因RGD-IL-24重组腺病毒的构建及鉴定

杨珺¹, 骆菁菁², 胡秀芳²

¹浙江医学高等专科学校,杭州 310053,浙江;
²浙江理工大学生命科学学院,杭州 310018,浙江

收稿日期:2010-07-17 修回日期:2010-11-19 出版日期:2010-11-26 发布日期:2020-09-16
作者简介:杨珺,女,博士,副教授,研究方向:生物制药。Tel: 0571-87692692 E-mail: jennyyangjun@sina.com
基金资助:
浙江省"生物医学工程"重中之重学科开放基金(SWYX916);校级博士基金(2009D10923)

Construction of recombinant adenovirus containing tumor -targeting fusion RGD-IL-24 gene and analysis of its activity in vitro

YANG Jun¹, LUO Jing-jing², HU Xiu-fang²

¹Zhejiang Medical College, Hangzhou 310053,Zhejiang,China;
²School of Life Sciences, Zhejiang Sci-Tech University,Hangzhou 310018, Zhejiang, China

Received:2010-07-17 Revised:2010-11-19 Online:2010-11-26 Published:2020-09-16

摘要/Abstract

摘要： 目的: 构建靶向性抗肿瘤融合基因RGD-IL-24的重组腺病毒载体,转染乳腺癌MCF-7细胞观察其感染,并对其体外抗肿瘤效应和肿瘤靶向性进行初步研究。方法: 利用PCR技术将GRGDS序列融合至IL-24的N端,克隆至pAdTrack-CMV质粒上。经PmeI线性化后与腺病毒骨架pAdEasy-1共转化大肠杆菌BJ5183,通过细菌内同源重组获得重组腺病毒载体pAdEasy/RGD-IL-24。重组腺病毒载体经PacI线性化后转染293细胞,包装出重组腺病毒Ad.RGD-IL-24。PCR方法鉴定重组腺病毒,TCID₅₀测定病毒滴度,MTT法和形态学分析Ad.RGD-IL-24诱导MCF-7乳腺癌细胞凋亡的活性,细胞黏附实验评价其肿瘤靶向性。结果: 成功构建腺病毒载体,腺病毒滴度达 1.3×10⁹ pfu／mL。Ad.RGD-IL-24诱导乳腺癌MCF-7细胞凋亡,显著抑制其生长,并具有肿瘤靶向性。结论: 成功构建了有较强感染能力和肿瘤靶向性的重组RGD-IL-24腺病毒载体。

关键词: RGD, 白介素-24, 腺病毒, 肿瘤靶向性, 抗肿瘤活性

Abstract: AIM: To construct the recombinant adenovirus encoding tumor-targeting fusion RGD-IL-24 gene, and to study its biological activity in vitro. METHODS: The fusion gene GRGDS fused with N-terminus of IL-24 was obtained by PCR. Then it was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. The recombinant adenovirus was transfected into 293 cells using Lipofectine DOTAP.The target gene was detected by PCR. The titer and its infection rate were determined using the green fluorescent protein(GFP) expression in the shuttle plasmid.The activity of inducing tumor cells apoptosis was confirmed by MTT assay and Hoechst assay in vitro. RESULTS: The recombinant adenovirus vector was successfully constructed. The titer of the recombinant adenovirus was 1.3×10¹² pfu/mL. Ad.RGD-IL-24 induced apoptosis of MCF-7 cells, and RGD-IL-24 was targeted to tumor cells in vitro. CONCLUSION: The recombinant adenovirus containing RGD-IL-24 gene is successfully constructed by the method of homogenous recombination in bacteria.

Key words: Interleukin-24, Adenovirus, Tumor targeting, Anti-tumor activity

中图分类号:

杨珺, 骆菁菁, 胡秀芳. 靶向性抗肿瘤融合基因RGD-IL-24重组腺病毒的构建及鉴定[J]. 中国临床药理学与治疗学, 2010, 15(11): 1223-1228.

YANG Jun, LUO Jing-jing, HU Xiu-fang. Construction of recombinant adenovirus containing tumor -targeting fusion RGD-IL-24 gene and analysis of its activity in vitro[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2010, 15(11): 1223-1228.

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