内皮抑素重组腺病毒载体的构建及制备  

Construction of Recombinant Adenovirus Vector Expressing the Mouse Endostatin

作　　者：张子祥[1] 李德春[1] 赵华[1] 朱兴国[1] 

机构地区：[1]苏州大学附属第一医院普外科,江苏苏州215006

出　　处：《苏州大学学报（医学版）》2004年第5期654-656,680,共4页Suzhou University Journal of Medical Science

基　　金：卫生部科学研究基金资助课题(WKJ2004-2-001)

摘　　要：目的 构建鼠源性内皮抑素(mEndostatin)重组腺病毒真核表达载体,为下一步研究其在体外表达及动物实验研究提供基础。方法 以mEndostatin质粒为模板通过PCR方法扩增回收mEndostatin,并在其5'端引入M-成瘤蛋白信号肽。将mEndostatin连接到腺病毒载体pCA13中,构建pCA13-mEndo表达质粒。将此表达质粒与腺病毒重组质粒pBGHE3通过Lipofectamine 2000共同转染293细胞,经同源重组产生重组腺病毒Ad-mEndo。纯化后重组腺病毒Ad-mEndo在293细胞大量扩增并通过氯化铯密度梯度离心法纯化、测定其滴度。结果 扩增得到的mEndostatin经酶切鉴定、PCR扩增、DNA测序证实为鼠源性内皮抑素,重组腺病毒Ad-mEndo的滴度为6.3×1010 pfu／ml。结论 该实验为今后研究重组腺病毒mEndostatin用于恶性肿瘤的基因治疗打下了基础。Objective To construct the recombinant adenovirus vector expressing the mouse endo-statin and provide the basis for following experiments in vivo and in vitro. Methods The mEndostatin DNA which was extracted from vector containing mouse endostatin gene was successfully amplified by using PCR and then cloned into adenovirus vector pCA13. Recombinant adenovirus plasmid pCA13-mEndo was co-transfected with pBHGS into 293 packaging cells by lipofectamine2000 and the replication-deficient recombinant adenovirus Ad-mEndo was generated efficiently by homologous recombination. The Ad-mEndo recombinant adenovirus was efficiently duplicated in 293 cells and was purified by CsCl density centrifugation and the titer was measured. Results The mEndostatin cDNA was confirmed by sequencing. The virus titer was 6.3×1010pfu/ml. Conclusion This investigation provides the basis for studying tumor gene therapy by endostatin.

关 键 词：内皮抑素 腺病毒 真核表达 

分 类 号：R394.8[医药卫生—医学遗传学]