| Ad-EGFP-MCP-1腺病毒表达载体的构建与表达 |
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| 引用本文： | 张 芳,王 伟,谢 悦,张 昊. Ad-EGFP-MCP-1腺病毒表达载体的构建与表达[J]. 中国组织工程研究, 2011, 15(50): 9428-943. DOI: 10.3969/j.issn.1673-8225.2011.50.029 |
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| 作者姓名： | 张 芳 王 伟 谢 悦 张 昊 |
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| 作者单位： | 1江苏省中医药研究院风湿免疫科,江苏省南京市 2100282徐州医学院附属医院心胸外科,江苏省徐州市 221002 |
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| 基金项目： | 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。 |
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| 摘 要： | 背景：单核细胞趋化因子(monocyte chemotactic protein, 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pBHG lox ΔE1,3 Cre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。
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| 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 |
| 收稿时间： | 2011-05-19 |
Construction and expression of Ad-EGFP-MCP-1 vector |
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| Zhang Fang,Wang Wei,Xie Yue,Zhang Hao. Construction and expression of Ad-EGFP-MCP-1 vector[J]. Chinese Journal of Tissue Engineering Research, 2011, 15(50): 9428-943. DOI: 10.3969/j.issn.1673-8225.2011.50.029 |
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| Authors: | Zhang Fang Wang Wei Xie Yue Zhang Hao |
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| Affiliation: | 1Department of Rheumatology and Clinical Immunology, Jiangsu Provincial Institute of Traditional Chinese Medicine, Nanjing 210028, Jiangsu Province, China
2Department of Cardiothoracic Surgery, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China |
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| Abstract: | 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 titer 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 lox ΔE1,3 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. |
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