Ad-EGFP-MCP-1腺病毒表达载体的构建与表达  

Construction and expression of Ad-EGFP-MCP-1 vector

作　　者：张芳[1] 王伟[2] 谢悦[2] 张昊[2] 

机构地区：[1]江苏省中医药研究院风湿免疫科,江苏省南京市210028 [2]徐州医学院附属医院心胸外科,江苏省徐州市221002

出　　处：《中国组织工程研究与临床康复》2011年第50期9428-9432,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30571845),课题名称:HGF基因转染对高肺血流所致肺动脉高压的影响;江苏省自然科学基金资助项目(BK2007033),课题名称:序贯调控VEGF/ANG-1基因诱导缺血心肌新生血管生成作用机制的研究;徐州市科技计划基金资助项目(XM09B072),课题名称:HGF基因转染诱导肺血管生成治疗结缔组织病相关肺动脉高压~~

摘　　要：背景:单核细胞趋化因子(monocyte chemotacti cprotein,MCP)可促进新生血管的动脉化,形成功能性微动脉,进而实现血管新生。目的:构建Ad-EGFP-MCP-1腺病毒表达载体,包装、纯化并检测病毒滴度,观察其在体表达。方法:PCR法钓取目的基因MCP-1,将其亚克隆入pDC315-EGFP载体,利用辅助包装质粒pBHGloxΔE1,3Cre进行病毒包装得到Ad-EGFP-MCP-1,经呼吸道途径转染至肺动脉高压大鼠。结果与结论:通过PCR获得MCP-1全长cDNA,与GenBank比对,序列完全一致。荧光显微镜下可见所包装的Ad-EGFP-MCP-1腺病毒表达载体可在大鼠肺组织中稳定表达,1周时达高峰,持续约4周。证实实验成功构建并包装了Ad-EGFP-MCP-1,其在肺组织中可有效表达。BACKGROUND： Monocyte chemotactic factor can promote arterialization of newly formed vessels to form functional arteriole and then realize angiogenesis. OBJECTIVE： To construct Ad-EGFP-MCP-1 vector and to observe its expression in vivo after package, purification and viral liter detection. METHODS： MCP-1 gene was amplified by PCR and the sequence was compared with Genebank data, and then sub-cloned into the pDC315-EGFP vector after sequence analysis. Ad-EGFP-MCP-1 was obtained with pBHG Iox AE1,3 Cre secondary packaging system. After transfection of Ad-EGFP-MCP-1 via trachea, EGFP expression was observed under fluorescence microscope. RESULTS AND CONCLUSION： The full-length of MCP-1 was obtained by PCR and identified by sequencing. Ad-EGFP-MCP-1 recombinant adenovirus vector was stably expressed in rat lung tissue. EGFP expression reached the peak level at 1 week and maintained this level for approximately 4 weeks. Ad-EGFP-MCP-1 recombinant adenovirus vector was successfully constructed, packaged and amplified, and it is effectively expressed in the lung tissue.

关 键 词：单核细胞趋化因子1 腺病毒表达载体 肺动脉高压 构建 组织工程 

分 类 号：R318[医药卫生—生物医学工程]