An Improved Technique of Construction of Recombinant Adeno-Associated Virus 2 Mediated Gene Transfer to TIMP1 and Identification_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LI Minghao ¹ , WU Xiaobing ² , YAN Lnan ³

1.Department of Hepatobiliary Surgery, Affiliated Hospital of Ningxia Medical College, Yinchuan 750004, China;;
2.State Key Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052, China;;
3.Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, China;

Corresponding author：

Keywords：

Recombinant adeno-associated virus 2; Tissue inhibitor of metalloproteinase-1; Hepatocellular carcinoma; Gene therapy

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Objective To use an improved technique to construct the recombinant adeno-associated virus 2 (rAAV2) mediated gene which can transfer human tissue inhibitor of metalloproteinase-1 (TIMP1). Methods Human TIMP1 gene was amplified from pDNR-LIB plasmid by PCR and cloned into the rAAV2 vector pSNAV to recombinant pSNAV-TIMP1, then was transferred into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell named BHK-21/rAAV2-TIMP1 was isolated, which was capable to express TIMP1. The cell was subsequently infected with recombinant herpes simplex virus 1 (rHSV1-rc/△UL2) that was able to package the rAAV2-TIMP1. After purification, rAAV2-TIMP1 was obtained. Results The rAAV2 carrying human TIMP1 gene was constructed successfully. The viral titer of the rAAV2-TIMP1 was 1×1012 v.g./ml. Conclusion rAAV2-TIMP1 was constructed successfully, which would provide experimental basis for carrying the TIMP1 into hepatocellular carcinoma effectively and inhibiting the invasiveness and migratory capacity of hepatocellular carcinoma in vitro and in vivo models.

Citation： LI Minghao,WU Xiaobing,YAN Lnan. An Improved Technique of Construction of Recombinant Adeno-Associated Virus 2 Mediated Gene Transfer to TIMP1 and Identification. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2008, 15(12): 898-902. doi: Copy

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13.	Hermonat PL, Quirk JG, Bishop BM, et al. The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vectors [J]. FEBS Lett, 1997; 407(1)∶78.
14.	Phillips MI. Antisense inhibition and adeno-associated viral vector delivery for reducing hypertension [J]. Hypertension, 1997; 29(1 Pt 2)∶177.
15.	Grimm D, Kern A, Rittner K, et al. Novel tools for production and purification of recombinant adenoassociated virus vectors [J]. Hum Gene Ther, 1998; 9(18)∶2745.
16.	Zolotukhin S, Byrne BJ, Mason E, et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield [J]. Gene Ther, 1999; 6(6)∶973.
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19.	伍志坚, 吴小兵, 侯云德. 系列腺病毒伴随病毒载体的构建及表达β-半乳糖苷酶的研究 [J]. 病毒学报， 2000; 16(1)∶1.

1. Jiang Y, Goldberg ID, Shi YE. Complex roles of tissue inhibitors of metalloproteinases in cancer[J].Oncogene, 2002; 21(14)∶2245.
2. 赵新，　张培瑞，　俞祥海，　等. 　腺病毒介导TIMP2基因转染抑制大鼠腹主动脉细胞外基质降解的实验研究　[J].　中国普外基础与临床杂志, 2007; 14（3）∶276.
3. Jia L, Zhou H, Wang S, et al. Deglycosylation of CD147 down-regulates Matrix Metalloproteinase-11 expression and the adhesive capability of murine hepatocarcinoma cell HcaF in vitro [J]. IUBMB Life, 2006; 58(4)∶209.
4. Sambrook J, Fritsch EF, Maniatis T Eds. Molecular cloning: A Laboratory Manual[M]. 2nd ed. New York: CHS press, 1989.
5. 伍志坚，吴小兵，曹晖，等. 一种高效的重组腺伴随病毒载体生产系统 [J]. 中国科学(C辑), 2001; 31(5)∶423.
6. 吴小兵，董小岩，伍志坚，等. 一种快速高效分离和纯化腺病毒伴随病毒载体的方法 [J]. 科学通报， 2000; 45(19)∶2071.
7. Debelak D, Fisher J, Iuliano S, et al. Cation-exchange high-performance liquid chromatography of recombinant adeno-associated virus type 2[J]. J Chromatogr B Biomed Sci Appl, 2000; 740(2)∶195.
8. Matsumoto E, Nakatsukasa H, Nouso K, et al. Increased levels of tissue inhibitor of metalloproteinase-1 in human hepatocellular carcinoma [J]. Liver Int, 2004; 24(4)∶379.
9. Monahan PE, Samulski RJ, Tazelaar J, et al. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia [J]. Gene Ther, 1998; 5(1)∶40.
10. Rabinowitz JE, Samulski RJ. Building a better vector: the manipulation of AAV virions[J]. Virology, 2000; 278(2)∶30111 Santat L, Paz H, Wong C, et al. Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice [J]. Proc Natl Acad Sci USA, 2005; 102(31)∶11053.
11. 刘自明，严律南，李馨筱. 包载携有HSVTK基因腺病毒PLG微球的制备 [J]. 中国普外基础与临床杂志, 2007; 14（4）∶399.
12. 张明满，李德华，苟兴华, 等. 靶向肝癌细胞的重组腺相关病毒载体的构建 [J]. 中国普外基础与临床杂志, 2005; 12(2)∶142.
13. Hermonat PL, Quirk JG, Bishop BM, et al. The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vectors [J]. FEBS Lett, 1997; 407(1)∶78.
14. Phillips MI. Antisense inhibition and adeno-associated viral vector delivery for reducing hypertension [J]. Hypertension, 1997; 29(1 Pt 2)∶177.
15. Grimm D, Kern A, Rittner K, et al. Novel tools for production and purification of recombinant adenoassociated virus vectors [J]. Hum Gene Ther, 1998; 9(18)∶2745.
16. Zolotukhin S, Byrne BJ, Mason E, et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield [J]. Gene Ther, 1999; 6(6)∶973.
17. Clark KR, Liu X, McGrath JP, et al. Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses [J]. Hum Gene Ther, 1999; 10(6)∶1031.
18. 夏冬，徐亮，万礼仪，等. 重组腺病毒介导hTIMP1对人肝癌细胞系体外增殖的影响 [J]. 中国普外基础与临床杂志, 2007; 14（4）∶409.
19. 伍志坚, 吴小兵, 侯云德. 系列腺病毒伴随病毒载体的构建及表达β-半乳糖苷酶的研究 [J]. 病毒学报， 2000; 16(1)∶1.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

An Improved Technique of Construction of Recombinant Adeno-Associated Virus 2 Mediated Gene Transfer to TIMP1 and Identification

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