肿瘤血管抑制肽alphastatin重组腺伴随病毒的制备  

Preparation of the antiangiogenic peptide alphastatin recombinant adeno-associated virus

作　　者：郭世文[1] 杨宇[2] 王全颖 廉民学[1] 李涛[1] 王东峰[1] 

机构地区：[1]西安交通大学医学院第一附属医院神经外科,陕西西安710061 [2]吉林大学第一医院神经内科,吉林长春130021 [3]西安华广生物工程有限公司,陕西西安710025

出　　处：《中华神经外科疾病研究杂志》2006年第6期518-521,共4页Chinese Journal of Neurosurgical Disease Research

基　　金：国家自然科学基金资助项目(30672162)

摘　　要：目的构建并产生肿瘤血管抑制肽alphastatin(Al)重组腺伴随病毒(rAAV)载体。方法将目的基因alphastatin插入载体质粒pSSHG-巨细胞病毒(CMV)的EcoRI和BamHI位点,构建重组质粒pSSHG-CMV/NT4-Al。用腺病毒辅助质粒pFG140代替野生型腺病毒,包装质粒pAAV/Ad及已构建的重组腺伴随病毒载体质粒,使用三质粒共转染法转染293细胞,包装得到腺伴随病毒(AAV)-Al。采用氯仿抽提、聚乙二醇沉淀回收纯化,斑点杂交方法测定重组病毒滴度。结果重组病毒rAAV-Al滴度约为2×1012颗粒/ml。结论成功制备了重组病毒rAAV-Al,提供了一种生产足量安全的rAAV-Al简单易行的方法,为肿瘤的基因治疗实验奠定了基础。Objective To construct and produce the vector of antiangiogenic peptide alphastatin （M） recombinant adeno-associated virus （rAAV）. Methods By inserting the resulting gene of Al into the EcoR Ⅰ and BamH Ⅰ sites of vector plasmid pSSHG- cytomegalovirus （ CMV）, the recombinant plasmid vector pSSHG-CMV/ NT4-Al was constructed. The 293 cells were co-transfected with the recombinant plasmid, packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140 instead of wild type adenovirus. The rAAV-Al viral stock was purified by chloroform and polyethylene glycol precipitation and titrated by dot blot. Results The titer of rAAV- Al vector was approximately 2 × 10^12 particles/ml. Conclusion The viral stock of rAAV-M is successfully prepared, which can lay a basis for the experimental studies of gene therapy of tumors.

关 键 词：alphastatin 腺伴随病毒 斑点杂交 

分 类 号：Q78[生物学—分子生物学]