[1]王峰,许瑞安.表达Kallistatin的9型重组腺相关病毒的制备及其体外表达效率[J].华侨大学学报(自然科学版),2013,34(1):68-71.[doi:10.11830/ISSN.1000-5013.2013.01.0068]
　WANG Feng,XU Rui-an.Production and Expression Efficiency in Vitro of Recombinant Adeno-Associated Virus 9 Mediating Kallistatin[J].Journal of Huaqiao University(Natural Science),2013,34(1):68-71.[doi:10.11830/ISSN.1000-5013.2013.01.0068]
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表达Kallistatin的9型重组腺相关病毒的制备及其体外表达效率()
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《华侨大学学报（自然科学版）》[ISSN:1000-5013/CN:35-1079/N]

卷:
第34卷
期数:
2013年第1期
页码:
68-71
栏目:
出版日期:
2013-01-20

文章信息/Info

Title:
Production and Expression Efficiency in Vitro of Recombinant Adeno-Associated Virus 9 Mediating Kallistatin
文章编号:
1000-5013(2013)01-0068-04
作者:
王峰1; 2;  许瑞安1; 2
1. 华侨大学 分子药物研究院, 福建 泉州 362021;2. 华侨大学 分子药物教育部工程研究中心, 福建 泉州 362021
Author(s):
WANG Feng1; 2;  XU Rui-an1; 2
1. Institute of Molecular Medicine, Huaqiao University, Quanzhou 362021, China; 2. Engineering Research Center of Molecular Medicine, Ministry of Education, Huaqiao University, Quanzhou 362021, China
关键词:
9型重组腺相关病毒;  Kallistatin;  表达效率;  人脐静脉血管内皮细胞
Keywords:
recombinant adeno-associated virus type 9;  Kallistatin;  expression efficiency;  HUVEC
分类号:
R969.4
DOI:
10.11830/ISSN.1000-5013.2013.01.0068
文献标志码:
A
摘要:
采用三质粒磷酸钙共转染生产系统,制备出可以表达内源性抗肿瘤血管生成因子(Kallistatin)的9型重组腺相关病毒(rAAV2/9)和2型重组腺相关病毒(rAAV2/2).rAAV经银染法鉴定其纯度;采用Western Blotting法比较rAAV2/9和rAAV2/2外壳蛋白的大小;qPCR方法检测rAAV的滴度.使用rAAV2/9-Kallistatin和rAAV2/2-Kallistatin分别感染人脐静脉血管内皮细胞(HUVEC)后,RT-PCR和ELISA法分别检测Kallistatin的mRNA和蛋白表达水平.结果表明:纯化的病毒纯度达到98%以上;rAAV9外壳蛋白比rAAV2的大.感染HUVEC后, rAAV2/9介导Kallistatin在mRNA和蛋白表达水平都要比rAAV2/2高.
Abstract:
Recombinant adeno-associated virus 9(rAAV2/9)and recombinant adeno-associated virus 2(rAAV2/2)had been produced by three plasmids packaging system. rAAV2/9 and rAAV2/2 could express Kallistatin that had the activity of anti-angiogenesis. The purity of rAAV was detected by Silver stain. The molecular weight of rAAV2/9 and rAAV2/2 were detected by Western Blotting. The titers of rAAV were detected by real-time quantitative PCR(qPCR). After rAAV2/9- or rAAV2/2-Kallistatin infecting human umbilical vein endothelial cell(HUVEC), the expression levels of mRNA and protein were detected by reverse transcription PCR(RT-PCR)and enzyme-linked immunosorbnent assay(ELISA)respectively. The results indicated that the purity of rAAV was more than 98% and molecular weight of rAAV2/9 was more than rAAV2/2’s. After infecting HUVEC, the expression levels of mRNA and protein of Kallistatin mediated by rAAV2/9 were higher than rAAV2/2’s.

参考文献/References:

[1] XU Rui-an,SUN Xue-ying,TSE Lai-yin,et al.Long-term expression of angiostatin suppresses metastatic liver cancer in mice[J].Hepatology,2003,37(6):1451-1460.
[2] 舒静波,刁勇,肖卫东,等. 腺病毒-腺相关病毒嵌合载体的研发趋势[J]. 华侨大学学报:自然科学版,2008,29(2):172-176.
[3] 王峰,刁勇,肖卫东,等.重组腺相关病毒规模化生物包装技术[J].生物工程学报,2009,25(11):1608-1613.
[4] MAGUIRE A M,SIMONELLI F,PIERCE E A, et al.Safety and efficacy of gene transfer for Leber’s congenital amaurosis[J].N Engl J Med,2008,358(21):2240-2248.
[5] GAO Guang-ping,VANDENBERGHE L H,ALVIRA M R,et al.Clades of adeno-associated viruses are widely disseminated in human tissues[J].J Virol,2004,78(12):6381-6388.
[6] 许瑞安,陈凌,肖卫东.分子基因药物学[M].北京:北京大学出版社,北京大学医学出版社,2008:26-32.
[7] QI Yan-fei,LIU Xuan,LI Hong-wei,et al.Selective tropism of the recombinant adeno-associated virus 9 serotype for rat cardiac tissue[J].J Gene Med,2010,12(1):22-34.
[8] LIMBERIS M P,WILSON J M,Adeno-associated virus serotype 9 vectors transduce murine alveolar and nasal epithelia and can be readministered[J].Proc Natl Acad Sci USA,2006,103(44):12993-12998.
[9] MIAO R Q,AGATA J,CHAO L,et al.Kallistatin is a new inhibitor of angiogenesis and tumor growth[J].Blood,2002,100(9):3245-3252.
[10] TSE Lai-yin,SUN Xue-ying,JIANG Hong-chi,et al.Adeno-associated virus-mediated expression of kallistatin suppresses local and remote hepatocellular carcinomas[J].J Gene Med,2008,10(5):508-517.
[11] XU R,RAHIMI M,MA H,et al.Stability of infectious recombinant adeno-associated viral vector in gene delivery[J].Med Sci Monit,2005,11(9):R305-R308.
[12] GRIEGER J C,CHOI V W,SAMULSKI R J,Production and characterization of adeno-associated viral vectors[J].Nat Protoc,2006,1(3):1412-1428.
[13] MITCHELL M,NAM H J,CARTER A,et al.Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9[J].Acta Crystallogr F,2009,65:715-718.
[14] BOUTIN S,MONTEILHET V,VERON P,et al.Prevalence of serum IgG and neutralizing factors against adeno-associated virus(AAV)types 1,2,5,6,8, and 9 in the healthy population: Implications for gene therapy using AAV vectors[J].Hum Gene Ther,2010,21(6):704-712.
[15] DIAO Yong,MA Jun,XIAO Wei-dong,et al.Inhibition of angiogenesis and Hg-116 xenograft tumor growth in mice by kallistatin[J].World J of Gastroenterol,2007,13(34):4615-4619.
[16] JRMSL A,BTSY F,CENTER M M,et al.Global cancer statistics[J].Cancer J Clin,2011,61(2):69-90.

备注/Memo

备注/Memo:
收稿日期: 2012-05-24
通信作者: 许瑞安(1948-),男,教授,主要从事分子药物、分子医学、海洋与基因工程药物的研究.E-mail:ruianxu@hqu.edu.cn.
基金项目: 国家自然科学基金资助项目(81072578); 福建省自然科学基金资助项目(2010J05026); 福建发改委项目(20090205); 福建省厦门市科技计划项目(3502Z20053046)
更新日期/Last Update: 2013-01-20