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目的:构建人血管内皮生长因子165(hVEGF165)的真核表达载体,并在大鼠骨髓基质细胞中进行表达。方法:利用基因克隆技术,将原核克隆载体pSP73中的目的基因VEGF165用 BamH I和Xho I双酶切后,再克隆到真核表达载体pcDNA3.1中,构建重组质粒pcDNA3.1-VEGF165。对重组质粒进行酶切分析和测序鉴定。通过脂质体介导,用重组质粒转染SD大鼠骨髓基质细胞,然后以G418筛选阳性克隆,用免疫细胞化学鉴定。结果:经酶切鉴定及基因测序证实,重组体中已插入目的基因片段VEGF165。免疫细胞化学证实,重组质粒转染的骨髓基质细胞中有VEGF165基因的表达。结论:成功地构建真核表达载体pcDNA3.1-VEGF165,并在骨髓基质细胞中得到表达,为将表达VEGF基因的骨髓基质细胞作为骨组织工程的种子细胞的可能性提供了实验依据。
Objective: To construct eukaryotic expression vector of human vascular endothelial growth factor 165 (hVEGF165) and express it in rat bone marrow stromal cells. Methods: The target gene VEGF165 in prokaryotic cloning vector pSP73 was digested with BamH I and Xho I by gene cloning technique, then cloned into eukaryotic expression vector pcDNA3.1 to construct recombinant plasmid pcDNA3.1-VEGF165. The recombinant plasmid was digested and sequenced. Mediated by liposome, SD rat bone marrow stromal cells were transfected with the recombinant plasmids, and positive clones were screened by G418 and identified by immunocytochemistry. Results: The result of restriction enzyme digestion and gene sequencing confirmed that the target gene fragment VEGF165 was inserted into the recombinants. Immunocytochemistry confirmed that the recombinant plasmid transfected bone marrow stromal cells have VEGF165 gene expression. CONCLUSION: The eukaryotic expression vector pcDNA3.1-VEGF165 was successfully constructed and expressed in bone marrow stromal cells, providing an experimental basis for the possibility of using bone marrow stromal cells expressing VEGF gene as a seed tissue for bone tissue engineering.