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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2011, Vol. 31 Issue (06): 86-92    
技术与方法     
腺病毒载体纤突结的改造及改造后的特性研究
乔伟1,2, 何金生1,2, 付远辉2, 洪涛2,3
1. 安徽医科大学免疫学教研室 合肥 230032;
2. 北京交通大学生命科学与生物工程研究院 北京 100044;
3. 中国疾病预防与控制中心病毒病预防控制所 北京 100052
The Characterization of Modified Adenovirus Vector
QIAO Wei1,2, HE Jin-shen1,2, FU Yuan-hui2, HONG Tao2,3
1. Department of Immunology, Anhui Medical University, Hefei 230032, China;
2. College of Life Sciences & Bioengineering, Beijing Jiaotong University, Beijing 100044, China;
3. Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China
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摘要:

目的:采用一种简便方法在复制缺陷型腺病毒载体纤突结(fibre knob)的HI loop插入精氨酸-甘氨酸-天冬氨酸(arginine-glycine-aspartate,RGD)肽,构建纤突结修饰的腺病毒载体,观察其对RD和T24肿瘤细胞的感染效率以及机体针对外源基因的抗体反应。方法:以PCR方法将RGD插入到pAdEasy-1后,通过常规方法构建可表达EGFP和人呼吸道合胞病毒(human respiratory syncytial viruse, RSV)密码子优化型融合蛋白(fusion protein, Fsyn)的腺病毒载体FGAd-RGD-EGFP和FGAd-RGD-Fsyn。用FGAd-RGD-EGFP感染RD和T24肿瘤细胞,通过荧光显微镜和流式细胞仪检测感染效率,用FGAd-RGD-Fsyn免疫BALB/c小鼠,通过ELISA检测针对外源基因的抗体反应。结果:与FGAd-EGFP相比,FGAd-RGD-EGFP对RD和T24肿瘤细胞的感染效率都有显著提高,EGFP表达效率也明显增强。与FGAd-Fsyn相比,FGAd-RGD-Fsyn所引起的机体针对外源基因的抗体反应并无差异。结论:改造后的腺病毒载体能显著提高对RD和T24肿瘤细胞的感染效率,但并不引起机体产生针对外源基因的增强的抗体反应,该载体能用于肿瘤等疾病的基因治疗研究。

关键词: 复制缺陷型腺病毒载体RGD荧光抗体反应    
Abstract:

Objective: A simple way to construct an adenovirus vector containing a peptide-RGD in the HI loop of the fiber knob was developed and then its infection efficiency on RD and T24 cells in vitro and the humoral responses to transgenes in vivo were investigated. Methods: The pAdEasy-RGD-1 was modified by inserting RGD motif into pAdEasy-1 based on simple PCR method. Enhanced green fluorescent protein (EGFP) and codon optimized fusion protein (Fsyn) of human respiratory syncytial virus (RSV) were cloned and constucted into pAdEasy-RGD-1 to generate adenovirus vectors of FGAd-RGD-EGFP and FGAd-RGD-Fsyn, respectively. FGAd-RGD-EGFP infected RD and T24 cells to investigate its infection efficiency using fluorescent microscope and flow cytometry. FGAd-RGD-Fsyn was exploited to vaccinate BALB/c mice via intramuscular and imtranasal routes, respectively, to evaluate humoral responses to transgenes in vivo. Results: The infection efficiency on RD and T24 cells was higher with FGAd-RGD-EGFP than FGAd-EGFP, and immunization with FGAd-RGD-Fsyn generated similar serum IgG specific for RSV-F. Conclusions: PCR is a simple and practical method to construct adenovirus vector containing a peptide-RGD in the HI loop of the fiber knob. The resultant modified adenovirus vector displays improved infection efficiency on RD and T24 cells and similar humoral immune responses to transgene as compared to original adenovirus vector. Therefore, it will be an useful platform for oncotherapy.

Key words: Replication-defective adenovirus vector    RGD    Fluorensence    Humoral immune response
收稿日期: 2010-11-15 出版日期: 2011-06-28
ZTFLH:  Q819  
通讯作者: 何金生     E-mail: jshhe@bjtu.edu.cn
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引用本文:

乔伟, 何金生, 付远辉, 洪涛. 腺病毒载体纤突结的改造及改造后的特性研究[J]. 中国生物工程杂志, 2011, 31(06): 86-92.

QIAO Wei, HE Jin-shen, FU Yuan-hui, HONG Tao. The Characterization of Modified Adenovirus Vector. China Biotechnology, 2011, 31(06): 86-92.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I06/86

[1] Bergelson J M, Cunningham J A, Droguett G, et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science, 1997,275:1320-1323.
[2] Tomko R, Xu R, Philipson L. HCAR and MCAR: The human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci USA, 1997,94:3352-3356.
[3] Henry L J, Xia D, Wilke M E, et al. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli. J Virol, 1994,68:5239-5246.
[4] Nemerow G. Cell receptors involved in adenovirus entry. Virology,2000,274:1-4.
[5] Bai M, Harfe B, Freimuth P. Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells. J Virol, 1993,67:5198-5205.
[6] Wickham T, Mathias P, Cheresh D, et al. Integrins alpha V Beta3 and alpha V Beta5 promote adenovirus internalization but not virus attachment. Cell, 1993,73:309-319.
[7] Li Y, Pong R, Bergelson J, et al. Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res, 1999,59:325-330.
[8] Cripe T, Dunphy E, Holub A, et al. Fiber knob modifications overcome low, heterogeneous expression of the Coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells. Cancer Res, 2001,61:2953-2960.
[9] Asaoka K, Tada M, Sawamura Y, et al. Dependence of efficient adenoviral gene delivery in malignant glioma cells on the expression levels of the Coxsackievirus and adenovirus receptor. J Neurosurg, 2000,92:1002-1008.
[10] Naoki Okada,Yasushige Masunaga,Yuka Okada, et al. Gene transduction efficiency and maturation status in mouse bone marrow-derived dendritic cells infected with conventional or RGD fiber-mutant adenovirus vectors. Cancer Gene Therapy, 2003,10:421-431.
[11] Dmitriev I, Krasnykh V, Miller C R, et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a Coxsackievirus. J Virol, 1998,72:9706-9713.
[12] Krasnykh V, Dmitriev I, Mikheeva G, et al. Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob. J Virol, 1998,72:1844-1852.
[13] Bouri K, Feero W G, Myerburg M M, et al. Poly-lysine modification of adenoviral fiber protein enhances muscle cell transduction. Hum Gene Ther, 1999, 10:1633-1640.
[14] Gonzalez R, Vereecque R, Wickham T J, et al. Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein. Gene Therapy, 1999,6:314-320.
[15] Ng P, Graham F L. Methods in Molecular Medicine. Gene Therapy Protocols. 2nd ed. Totowa: Human Press Inc, 2002, 69: 409.
[16] Ternette N, Tippler B, Uberla K, et al. Immunogenicity and efficacy of codon-optimized DNA vaccines encoding the F-protein of respiratory syncytial virus. Vaccine, 2007,25:7271-7279.
[17] Mizuguchi H, Koizumi N, Hosono T, et al. A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob. Gene Therapy, 2001,8:730-735.
[18] Stefan Worgall, Annette Busch, Michael Rivara, et al. Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses. J Virol, 2004,5: 2572-2580.

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