hVEGF_(165)和嵌合水蛭肽融合基因的真核表达载体的构建及其表达  

Construction of a eukaryotic expression vector hVEGF_(165)-fused hirudin/pcDNA3.0 and its expression

作　　者：陈少萍[1] 沈雳[1] 秦永文[1] 蔡在龙[2] 

机构地区：[1]第二军医大学长海医院心内科,上海200433 [2]基础医学部生物化学与分子生物学教研室,上海200433

出　　处：《第二军医大学学报》2005年第9期983-987,共5页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30200281)

摘　　要：目的:克隆hVEGF165和嵌合水蛭肽(fused hirudin,FH)融合基因,构建hVEGF165-FH/pcDNA3.0基因表达载体,并在人内皮细胞株中表达。方法:分别通过PCR法扩增hVEGF165和引物退火法合成FH基因,将融合基因插入表达载体pcD-NA3.0,构建重组质粒hVEGF165-FH/pcDNA3.0。对重组质粒进行酶切鉴定和测序分析,将正确克隆的质粒分别作体外快速转录翻译鉴定,以及经脂质体介导转染人内皮细胞株,并对表达产物进行鉴定。结果:hVEGF165和FH成功融合,形成711 bp的目的片段,并成功构建重组质粒hVEGF165-FH/pcDNA3.0,重组质粒转染人内皮细胞株有hVEGF165-FH表达。结论:重组质粒hVEGF165-FH/pcDNA3.0在人内皮细胞株中得到表达,为其融合蛋白活性研究及基因治疗打下良好基础。Objective：To clone the fused gene of hVEGF165, and hirudin,construct a eukaryotic expression vector hVEGF165FH/pcDNA3.0 and express the vector in human endothelial cell strain. Methods： hVEGF165 cDNA fragment was amplified by PCR and fused hirudin peptide was synthesized by primer annealing. The fused hVEGF165 and hirudin gene was cloned into vector pcDNA3.0 to construct a new vector hVEGF165-FH/pcDNA3.0 and the recombinant plasmid was confirmed by restriction enzyme and auto sequencing. The correctly cloned plasmids were subjected to rapid in vitro transcription and translation identification. Liposome mediated, recombinant plasmids were used to transfect human endothelial cell strain and the products were identified. Results： hVEGF165-FH was successfully fused and formed a 711 bp target fragment. The recombinant vector hVEGF165-FH/pcDNA3.0 was successfully transformed into human endothelial cell strain which expressed the fusion protein. Conclusion： The recombinant vector hVEGF165-FH/pcDNA3.0 can express fused protein in human endothelial cell strain, which paves a way for further study on the activity of the fusion protein and gene therapy.

关 键 词：HVEGF165 水蛭肽 基因融合 基因表达 载体 

分 类 号：Q782[生物学—分子生物学]