重组人神经生长因子腺相关病毒载体的构建及病毒滴度的测定  

Construction of recombinant pAAV-NGF-β virus vector and assay of its titer

作　　者：刘相萍[1] 罗文娟[2] 杨堃[1] 隋爱华[1] 吕振华[1] 王传富[2] 

机构地区：[1]青岛大学医学院附属医院分子生物学研究室,山东省青岛市266003 [2]青岛大学医学院附属医院眼科,山东省青岛市266003

出　　处：《眼科新进展》2007年第10期728-731,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30572011)~~

摘　　要：目的克隆人神经生长因子-β(nerve growth factor-β,NGF-β)基因,重组于腺相关病毒(adeno-associated virus,AAV)载体,构建表达人NGF-β的重组腺相关病毒,检测滴度,为NGF-β的真核表达及临床应用提供实验基础。方法从正常人基因组中扩增出人NGF-β全长开放读框,将其克隆于pAAV-MCS载体,双酶切和测序鉴定重组病毒载体。将该质粒与腺相关病毒包装质粒(pAAV-RC)、腺相关病毒辅助质粒(pAAV-Helper)共转染AAV-293细胞,通过包装得到重组腺相关病毒并回收病毒原液。以pAAV-LacZ和pAAV-RC、pHelper共转染AAV-293细胞,重组病毒AAV-LacZ感染AAV-HT1080细胞,经对报告基因LacZ的X-Gal染色,通过蓝染的细胞计算病毒滴度。结果PCR产物经电泳证明大小正确,重组质粒pAAV-NGF-β经双酶切和测序证实NGF-β基因正确插入载体且序列正确。通过AAV-LacZ感染AAV-HT1080细胞后X-gal染色计数,测定重组腺相关病毒的滴度为10×109pfu.L-1。结论成功构建了表达人NGF-β基因的重组腺相关病毒载体,为进一步研究NGF-β基因治疗角膜病打下基础。Objective To clone human nerve growth factor-β （NGF-β） gene into adeno-associated virus （AAV） vector and construct recombinant PAAV- NGF-β virus vector,providing experimental basis for clinical application and expression of NGF-β. Methods The full-length open reading frame （ORF） of NGF-β was amplified by PCR according to sequence data of GenBank. PCR products were inserted into pAAV-MCS vector to construct recombinant pAAVNGF-vector. After restrictive endonucleosidase analysis and DNA sequencing, the packaging of AAV-NGF-β was based on pAAV-C, pAAV-RC and pHelper cotransfecting into AAV-293 cells. Stock solution of virus was retrieved. Meanwhile, pAAV-LacZ, pAAV-RC and pHelper were co-transfected into AAV-293 cells,too. The titer of recombinant AAV-NGF-β was calculated by means of cells X-Ga1 staining,that expressed the report gene LacZ. Results The results of double enzyme digestion and DNA sequencing of recombinant pAAV-NGF-β plasmid showed that human NGF-β ORF had been inserted into pAAV-MCS correctly. After recombinant and package in AAV-293 cells, the recombinant pAAV-NGF-β viral particles were harvested with the titers of 10×10^9 pfu · L^-1, Conclusion The NGF-β ORF is cloned integrally. Recombinant pAAV-NGF-β vector is constructed correctly,which will benefit to fitrther study on its proliferation effect to mammalian corncal endothelial cells and related cell line and tissues.

关 键 词：神经生长因子 基因克隆 腺相关病毒 病毒滴度 角膜 

分 类 号：Q939.4[生物学—微生物学] R772.2[医药卫生—眼科]