[含hVEGF165基因的真核表达载体在大鼠骨髓基质细胞中的构建与表达]

[Construction and expression of eukaryotic expression vector containing hVEGF165 gene in rat bone marrow stroma cell].

作者信息

Gao Quan-wen, Dong Shao-zhong, Chen Xi-zhe, Chen Yan, Yang Lian-jia

机构信息

Department of Oral Histology and Pathology, College of Stomatology, Fourth Military Medical University, Xi'an 710032, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2003 Jan;19(1):32-4.

PMID:15132900

Abstract

AIM

To construct the eukaryotic expression vector containing human vascular endothelial growth factor 165(VEGF165) gene and express it in rat bone marrow stroma cells(rMSCs).

METHODS

The recombinant plasmid pSP73-VEGF165 was digested with BamH I and Xho I. Then the hVEGF165 gene segment obtained was again cloned into pcDNA3.1 to construct recombinant eukaryotic expression vector pcDNA3.1-VEGF165. Then the recombinant vector was identified by enzyme digestion analysis and sequencing. The rMSCs were transformed by recombinant vector and positive clones were screened with G418. The expression of hVEGF165 gene in the transformed cells was detected by immunocytochemical staining.

RESULTS

Enzyme digestion analysis and sequencing showed that target gene had been cloned into recombinant vector. The expression of hVEGF165 gene in the transformed cells had been demonstrated by immunocytochemical staining.

CONCLUSION

The recombinant eukaryotic expression vector has been constructed and expressed successfully in the transformed cells. Therefore, it is possible to use the rMSCs expressing hVEGF165 gene as seed cells in the bone tissue-engineering.

摘要

目的

构建含人血管内皮生长因子165（VEGF165）基因的真核表达载体，并在大鼠骨髓基质细胞（rMSCs）中表达。

方法

用BamH I和Xho I酶切重组质粒pSP73-VEGF165。将获得的hVEGF165基因片段再次克隆到pcDNA3.1中，构建重组真核表达载体pcDNA3.1-VEGF165。然后通过酶切分析和测序对重组载体进行鉴定。用重组载体转染rMSCs，并用G418筛选阳性克隆。通过免疫细胞化学染色检测转染细胞中hVEGF165基因的表达。