改良法構建Ⅱ型重組腺相關病毒介導人TIMP1基因及鑒定_《中國普外基礎與臨床雜志》

作者：

李明皓 ¹ , 吳小兵 ² , 嚴律南 ³

1.寧夏醫學院附屬醫院肝膽外科（銀川 750004）;;
2.中國預防醫學科學院病毒學研究所病毒基因工程國家重點實驗室（北京 100052）;;
3.四川大學華西醫院普外科（成都 610041）;

關鍵詞：

Ⅱ型重組腺相關病毒金屬蛋白酶抑制劑1 肝細胞癌基因治療

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目的改良法構建攜帶金屬蛋白酶抑制劑1(TIMP1)基因的Ⅱ型重組腺相關病毒（rAAV2）。方法采用PCR法從pDNR-LIB質粒中擴增人全長TIMP1基因，利用基因重組的方法將TIMP1全長cDNA插入通用型ＡＡＶ2載體質粒pSNAV的多克隆位點，構建成pSNAV-TIMP1；用脂質體轉染的方法將重組質粒轉入BHK-21細胞中，G418細胞篩選得到轉入重組質粒并能表達目的基因的細胞系BHK-21/rAAV2-TIMP1; 用具有rAAV2包裝功能的重組Ⅰ型單純皰疹病毒（rHSV1-rc/△UL2）感染BHK-21/rAAV2-TIMP1,純化后得到rAAV2-TIMP1。結果用改良法成功構建rAAV2-TIMP1,病毒滴度達到1×1012 v.g./ml。結論成功構建rAAV2-TIMP1,為其進一步應用于基因治療的研究提供實驗基礎。

引用本文： 李明皓,吳小兵,嚴律南. 改良法構建Ⅱ型重組腺相關病毒介導人TIMP1基因及鑒定. 中國普外基礎與臨床雜志, 2008, 15(12): 898-902. doi: 復制

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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.
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，萬禮儀，等. 重組腺病毒介導hTIMP1對人肝癌細胞系體外增殖的影響 [J]. 中國普外基礎與臨床雜志, 2007; 14（4）∶409.
19.	伍志堅, 吳小兵, 侯云德. 系列腺病毒伴隨病毒載體的構建及表達β-半乳糖苷酶的研究 [J]. 病毒學報， 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. 趙新，　張培瑞，　俞祥海，　等. 　腺病毒介導TIMP2基因轉染抑制大鼠腹主動脈細胞外基質降解的實驗研究　[J].　中國普外基礎與臨床雜志, 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. 伍志堅，吳小兵，曹暉，等. 一種高效的重組腺伴隨病毒載體生產系統 [J]. 中國科學(C輯), 2001; 31(5)∶423.
6. 吳小兵，董小巖，伍志堅，等. 一種快速高效分離和純化腺病毒伴隨病毒載體的方法 [J]. 科學通報， 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. 劉自明，嚴律南，李馨筱. 包載攜有HSVTK基因腺病毒PLG微球的制備 [J]. 中國普外基礎與臨床雜志, 2007; 14（4）∶399.
12. 張明滿，李德華，茍興華, 等. 靶向肝癌細胞的重組腺相關病毒載體的構建 [J]. 中國普外基礎與臨床雜志, 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，萬禮儀，等. 重組腺病毒介導hTIMP1對人肝癌細胞系體外增殖的影響 [J]. 中國普外基礎與臨床雜志, 2007; 14（4）∶409.
19. 伍志堅, 吳小兵, 侯云德. 系列腺病毒伴隨病毒載體的構建及表達β-半乳糖苷酶的研究 [J]. 病毒學報， 2000; 16(1)∶1.

《中國普外基礎與臨床雜志》

改良法構建Ⅱ型重組腺相關病毒介導人TIMP1基因及鑒定

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《中國普外基礎與臨床雜志》

改良法構建Ⅱ型重組腺相關病毒介導人TIMP1基因及鑒定

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

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