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

Abstract

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

CLC Number:

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.

References

[1] Jiang H, Lin J, Su Z, et al. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression[J]. Oncogene,1995,11(12):2477-2486.
[2] Caudell EG, Mumm JB, Poindexter N, et al. The protein product of the tumor suppressor gene, melanoma differentiation-associated gene-7, exhibits immunostimulatory activity and is designated IL-24[J]. Immunol,2002,168(12):6041-6046.
[3] Su Z, Lebedeva IV, Sarkar D, et al. Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in a p53-independent manner[J]. Oncogene,2003, 22(8):1164-1180.
[4] Emdad L, Lebedeva IV, Su Z, et al. Melanoma differentiation associated gene-7/interleukin-24 reverses multidrug resistance in human colorectal cancer cells[J]. Mol Cancer Ther,2007,6(11):2985-2994.
[5] Tucker GC.1nhibitors of integrins[J]. Curr Opin Pharmacol,2002,2(4):394-402.
[6] Ruoslahti E. The RGD story : a personal account[J].Matrix Biol, 2003,22(6):459-465.
[7] 丁娅,张晓实,彭瑞清,等.重组人p53腺病毒注射液治疗晚期实体肿瘤的安全性和近期疗效评价[J].中国临床药理学与治疗学,2005,10(9):1025-1029.
[8] 杨珺,蔡绍皙,邹全明,等.人IL-24基因的克隆、表达、纯化及抗肿瘤活性研究[J]. 细胞与分子免疫学杂志,2005,21(6):693-696.
[9] 奥斯伯 FM,金斯顿 RE,塞得曼 JG,等.精编分子生物学实验指南[M].4版.北京:科学出版社,2005:407-409.
[10] Kawabe S, Nishikawa T, Munshi A, et al.Adenovirus-mediated mda-7 gene expression radiosensitizes non-small cell lung cancer cells via TP532 independent mechanisms[J].Mol Ther,2002,6(5):637-643.
[11] Buckley CD, Pilling D, Henriquez NV, et al. RGD peptides induce apoptosis by direct caspase-3 activation[J].Nature,1999,397(6719):534-539.
[12] Mizuguchi H, Koizumi N, Hosono T, et al.A simplified system for constructing recombinant adenoviral vectors containing beterologouns peptides in the HI loop of their fiber knob[J].GeneTher,2001,8(9):730-735.