改良法构建Ⅱ型重组腺相关病毒介导人TIMP1基因及鉴定  

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

作　　者：李明皓[1] 吴小兵[2] 严律南[3] 

机构地区：[1]宁夏医学院附属医院肝胆外科,银川750004 [2]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室,北京100052 [3]四川大学华西医院普外科,成都610041

出　　处：《中国普外基础与临床杂志》2008年第12期898-902,909,共6页Chinese Journal of Bases and Clinics In General Surgery

摘　　要：目的改良法构建携带金属蛋白酶抑制剂1(TIMP1)基因的Ⅱ型重组腺相关病毒(rAAV2)。方法采用PCR法从pDNR-LIB质粒中扩增人全长TIMP1基因,利用基因重组的方法将TIMP1全长cDNA插入通用型AAV2载体质粒pSNAV的多克隆位点,构建成pSNAV-TIMP1;用脂质体转染的方法将重组质粒转入BHK-21细胞中,G418细胞筛选得到转入重组质粒并能表达目的基因的细胞系BHK-21/rAAV2-TIMP1;用具有rAAV2包装功能的重组Ⅰ型单纯疱疹病毒(rHSV1-rc/△UL2)感染BHK-21/rAAV2-TIMP1,纯化后得到rAAV2-TIMP1。结果用改良法成功构建rAAV2-TIMP1,病毒滴度达到1×1012v.g./ml。结论成功构建rAAV2-TIMP1,为其进一步应用于基因治疗的研究提供实验基础。Objective To use an improved technique to construct the recombinant adeno-associated virus 2 （rAAV2） mediated gene which can transfer human tissue inhibitor of metalloproteinaseq （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 （rHSVI-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 × 10^12 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.

关 键 词：Ⅱ型重组腺相关病毒 金属蛋白酶抑制剂1 肝细胞癌 基因治疗 

分 类 号：R735.7[医药卫生—肿瘤] Q754[医药卫生—临床医学]