人脑源性神经营养因子重组腺伴随病毒的制备  

Preparation of the human brain-derived neurotrophic factor recombinant adeno-associated virus

作　　者：马东亮[1] 胡海涛[1] 王建明[2] 任惠民[1] 刘勇[1] 杨广笑 

机构地区：[1]西安交通大学医学院人体解剖学教研室,陕西西安710061 [2]西安交通大学第二医院眼科,陕西西安710004 [3]西安华广生物工程公司,陕西西安710025

出　　处：《西安交通大学学报（医学版）》2003年第5期431-433,437,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：陕西省自然科学基金项目 (No .2 0 0 1SM 66);西安交通大学医学院基金

摘　　要：目的　构建并产生人脑源性神经营养因子 (humanbrain derivedneurotrophicfactor ,hBDNF)重组腺伴随病毒(adeno associatedvirus,AAV)载体。方法　将目的基因hBDNF插入载体质粒pSSHG Neo的EcoRI BamHI位点 ,构建重组质粒pSSHG Neo/hBDNF。用腺病毒辅助质粒 pFG14 0代替野生型腺病毒 ,包装质粒 pAAV/Ad及已构建的重组质粒 ,三质粒磷酸钙共沉淀法转染 80 %融合的 14 3细胞系 ,包装AAV hBDNF。经蔗糖梯度离心法纯化、斑点杂交方法测定重组病毒滴度。结果　重组病毒AAV hBDNF滴度约为 4 .86× 10 14 颗粒·L-1。结论　成功制备了重组病毒AAV hBDNF 。Objective To construct and produce the vector of the human brain-derived neurotrophic factor (hBDNF) recombinant adeno-associated virus (AAV). Methods By inserting the resulting gene of hBDNF into the EcoRI-BamHI site of vector plasmid pSSHG-Neo, we constructed the recombinant vector of plasmid pSSHG-Neo/hBDNF. The recombinant AAV viral stock was packaged. 80% of confluent 143 cell lines were co-transfected with the recombinant plasmid, packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140 instead of adenovirus by calcium phosphate precipitation. The recombinant AAV viral stock was purified by sucrose gradients and titrated by dot blot. Results The titer of recombinant viral vector was approximately 4.86×10 14 particles·L -1. Conclusion We have successfully prepared the viral stock of AAV-hBDNF that can serve as the experimental study of gene therapy of neurodegenerative diseases.

关 键 词：人脑源性神经营养因子 腺伴随病毒 神经退行性疾病 基因治疗 

分 类 号：R741[医药卫生—神经病学与精神病学] R394[医药卫生—临床医学]