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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 cprotein (MCP) promotes arteriogenesis of new blood vessels and forms functional arterioles that lead to angiogenesis. OBJECTIVE: To construct Ad-EGFP-MCP-1 adenovirus expression vector, packaging, purifying and detecting virus titer, and to observe its expression in vivo. METHODS: The target gene MCP-1 was isolated by PCR and subcloned into pDC315-EGFP vector. Ad-EGFP-MCP-1 was obtained by virus packaging with the aid of packaging plasmid pBHGloxΔE1,3Cre and transfection to pulmonary hypertension mouse. RESULTS AND CONCLUSION: The full-length cDNA of MCP-1 was obtained by PCR and aligned with GenBank. The sequence was identical. Under the fluorescence microscope, the packaged Ad-EGFP-MCP-1 adenovirus expression vector was stably expressed in the lung tissue of rats, reaching a peak at 1 week for about 4 weeks. It was confirmed that the experiment successfully constructed and packaged Ad-EGFP-MCP-1, which can be effectively expressed in lung tissue.