人血小板衍化生长因子-B真核表达质粒的构建-东南大学学报医学版

人血小板衍化生长因子-B真核表达质粒的构建

单位：1.东南大学附属中大医院整形烧伤外科,江苏南京,210009； 2.第二军医大学长征医院整形外科,上海,200003

分类号：Q782

出版年·卷·期（页码）：2003·22·第三期（144-147）

摘要：

目的:构建含人血小板衍化生长因子-B(hPDGF-B)基因的真核表达质粒,为深入研究hPDGF-B在组织修复中的作用及其在皮肤组织工程中的应用提供物质基础.方法:从原代培养的人脐静脉内皮细胞中提取总RNA,通过逆转录-聚合酶链反应扩增出hPDGF-B目的DNA片段.通过连接酶连入真核质粒pcDNA3.1中,经大肠杆菌DH5α扩增和筛选,构建出重组真核质粒pcDNA3.1-hPDGF-B.结果:重组质粒通过BamHⅠ和HindⅢ酶切后,电泳图显示0.6kb的hPDGF-B的目的片段和5.5kb的载体片段,证明重组质粒连接正确.经测序显示核苷酸及相应氨基酸序列正确无误.结论:含hPDGF-B基因的真核表达质粒被成功构建.

Objective To investigate the function of human platelet derived growth factor B (hPDGF?B) in wound healing,and its application to the cutaneous tissue engineering,our group intended to construct eukaryotic expression plasmid of hPDGF?B.Methods The hPDGF?B cDNA was amplified from the total RNA of the primary cultured endothelial cells of human umbilical vein and cloned into the eukaryotic plasmid pcDNA3.1 and screened with Escherichia coli DH5 α .Results The new recombinant plasmid was digested with Bam HⅠ and Hind Ⅲ,and the electrophoresis of the digested products showed two fragments of 0.6?kb and 5.5?kb,which proved appropriate ligation of the recombinant plasmid.The sequence of nucleotide and the corresponding amino acid of the cloned fragment were identical to those in the previous report.Conclusion The pcDNA 3.1 ?hPDGF?B,an eukaryotic expression plasmid of hPDGF?B gene is constructed.

参考文献：

[1] Collins T, GINSBURG D, MEREMY M. Cultured human endothelial cells express platelet-derived growth factor B chain:cDNA cloning and structural analysis. 1985. doi:10.1038/316748a0
[2] 王忠彪, 孙逊, 李勇. PDGF-B基因表达及其对成纤维细胞增生和胶原合成的促进作用. 中华创伤杂志1998(4)
[3] BREITBART A S, MASON J, URMACHER C. Gene-enhanced tissue engineering:applications for wound healing using cultured dermal fibroblasts transduced retovirally with the PDGF-B gene. 1999. doi:10.1097/00000637-199912000-00009
[4] Eming S A, MEDALIE D A, TOMPKINS R G. Genetically modified human keratinocytes overexpressing PDGF-A enhance the performance of a composite skin graft. 1998(9). doi:10.1089/hum.1998.9.4-529
[5] Norton J, PEPLINSKI G R, TSUNG K. Expression of secreted platelet-derived growth factor-B by recombinant nonreplicating and noncytopathic vaccinia virus. 1996. doi:10.1097/00000658-199610000-00013
[6] Liechty K W, NESBIT M, Herlyn M. Adenoviral-mediated overexpression of platelet-derived growth factor-B corrects ischemic impaired wound healing. 1999. doi:10.1046/j.1523-1747.1999.00705.x
[7] DEGUCH I J, NAMBA T, HAMMADA H. Targeting endogenous platelet-derived growth factor B-chain by adenovirus-mediated gene transfer potently inhibits in vivo smooth muscle proliferation after arterial injury. 1999(6). doi:10.1038/sj.gt.3300918
[8] Doukas J, CHANDLER L A, GONZALEZ A M. Matrix immobilization enhanced the tissue repair activity of growth factor gene therapy vectors. 2001(12). doi:10.1089/104303401750148720

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录