| 大鼠bcl-x_L cDNA的克隆和高滴度重组bcl-x_L腺相关病毒的制备 |
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| 引用本文： | 何湘君,Kunlin JIN,Reed CLARK,Steven H.GRAHAM.大鼠bcl-x_L cDNA的克隆和高滴度重组bcl-x_L腺相关病毒的制备[J].中国生物化学与分子生物学报,2001,17(4):468-472. |
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| 作者姓名： | 何湘君 Kunlin JIN Reed CLARK Steven H.GRAHAM |
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| 作者单位： | 何湘君（北京大学人民医院中心实验室,北京,100044）;Kunlin JIN（Department of Neurology, University of Pittsburgh; Pittsburgh,
PA 15213, USA;）;Steven H. GRAHAM（Department of Neurology, University of Pittsburgh;
Pittsburgh, PA 15213, USA;）;Reed CLARK（Children' s Hospital, The Ohio State University, Columbus, OH
43205, USA） |
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| 摘 要： | 以RT PCR法从大鼠脑组织中克隆bcl XL 基因 ,将其定向插入带rep cap基因和neu基因的三功能腺相关病毒 (AAV)载体 ,转染HeLa细胞并以G4 18筛选 ,然后用腺病毒感染筛选后的细胞克隆 ,包装成重组腺相关病毒 .用带rep cap基因的C12细胞筛选和滴定产生重组腺相关病毒的细胞克隆 ,粗制细胞裂解物中病毒滴度最高只有 3× 10 5IU ml.从产病毒量较高的克隆大量制备重组病毒 ,经肝素柱高压液相亲和层析法纯化、浓缩病毒后获得了高达 4× 10 11IU ml的重组病毒 .为研究脑缺血动物模型中BCL XL 的抗脑细胞凋亡作用打下了基础
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| 关 键 词： | bcl-xL 腺相关病毒 病毒滴度 制备 |
| 收稿时间： | 2001-08-20 |
| 修稿时间： | 2000年10月31 |
Cloning of Rat bcl-x _L cDNA and Production of Large Amount of Highly Purified Recombinant bcl-x_L /AAV |
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| HE Xiang\|jun ,Kunlin JIN ,Reed CLARK ,Steven H.GRAHAM.Cloning of Rat bcl-x _L cDNA and Production of Large Amount of Highly Purified Recombinant bcl-x_L /AAV[J].Chinese Journal of Biochemistry and Molecular Biology,2001,17(4):468-472. |
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| Authors: | HE Xiang\|jun Kunlin JIN Reed CLARK Steven HGRAHAM |
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| Institution: | ( 1) Central Laboratory,People's Hospital,Peking University,Beijing 100044,China; 2) Department of Neurology,University of Pittsburgh,Pittsburgh,PA 15213, |
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| Abstract: | In order to study the anti\|apoptosis effect of BCL\|X L on brain cells after ischemia, bcl\|x L containing adeno associated virus (AAV) vectors were constructed and a large amount of purified rAAV was obtained. bcl\|x L cDNA was cloned from rat brain tissues by RT\|PCR and inserted into AAV vector which contain ed rep\|cap gene and neu gene.The triple function plasmid was transfected into HeLa cells and the G418 resistant cell clones were selected.After infection with adenovirus,the crude cell lysates were identified for their rAAV production on C12 cells.The highest viral production cell line was enlarged to obtain a large amount of recombinant viruses.BioCad Sprint high\|performance liquid chromatography with heparin affinity column was used to purify the viruses.The final titer reached 4×10 11 IU/ml. |
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| Keywords: | bcl\|x L adeno\|associated virus vector viral titer |
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