摘要
目的:构建并转染含绿色荧光蛋白(GFP)报告基因的重组人血小板衍化生长因子-BB(rhPDGF-BB)非融合型表达载体,为采用转基因工程化细胞修复糖尿病溃疡创面奠定基础。方法:从人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)中分离总RNA,行RT-PCR获得rhPDGF-BB cDNA,构建克隆质粒pMD19-T/rhPDGF-BB,经测序正确后,再将该目的片段与真核表达载体pSELECT-GFPzeo-mcs连接,转化E.coli DH5α感受态菌落筛选得到含GFP报告基因的非融合型表达载体,然后将其转染中国仓鼠卵巢细胞(CHO细胞)并用免疫组化染色法鉴定。结果:PCR获得长度为681bp的目的片段,经pSELECT-GFPzeo-mcs载体连接、筛选及序列分析后,证实所插入目的片段与GenBank检索的rhPDGF-BB cDNA序列(NM-033016)完全匹配。结论:成功构建了非融合型表达载体pSELECT-GFPzeo-mcs/rhPDGF-BB,并实现了rhPDGF-BB基因在CHO的表达。
Objective： To construct the non-fusion expression vector with green fluorescent protein and then transform it into Chinese hamster ovary cells. Methods： The encoding fragment of recombinant human PDGF-BB gene was obtained from a human umbilical vein endothelial cell （HUVEC） line mRNA by reverse transcripfion-polymerase chain reaction. The PCR amplified fragment of rhPDGF-BB gene was cloned into pMD19-T vector,then the gene was inserted into the vector pSELECT-GFPzeo-mcs after sequencing,which introduced into E.coli DH 5α competent cells in order to choose and sequence. The non-fusion expression vector was constructed. RhPDGF-BB gene was transfected into Chinese hamster ovary cells and identified by immunohistochemistry. Results： A 681 bp fragment was obtained by PER clone after sequencing. The results were matching to the cDNA of rhPDGF-BB,which accession of the gene bank was NM-033016. Conclusion： The pSELECT-GFPzeo-mcs/rhPDGF-BB non-fusion expression vector was constructed successfully and the transient expression of rhPDGF-BB gene in Chinese hamster ovary cells has been realized.
出处
《天津医药》
CAS
北大核心
2008年第7期486-488,I0001,共4页
Tianjin Medical Journal
基金
中国博士后科学基金项目(项目编号:20070410766)
天津市卫生局科技基金项目(项目编号:07kz47)
关键词
遗传载体
绿色荧光蛋白质类
仓鼠
卵巢
转染
genetic vectors green fluorescent proteins hamster ovary transfection
