【摘要】 目的 :用一种简便方法构建了含血小板第四因子 (PF4)cDNA的腺病毒载体 ,体外检测其抑制人脐静脉内皮细胞 (HUVEC)活性。方法 :将含有信号肽的PF4cDNA克隆至腺病毒穿梭质粒 pAdshuttle CMV中 ,形成转移质粒pAdshuttle/PF4,将之用PmeI酶切线性化后与腺病毒骨架质粒 pAdEasy 1共转化大肠杆菌BJ5 183 ,抽提并鉴定含目的基因的重组体质粒 ,PacI酶切后用磷酸钙共沉淀方法转染HEK2 93细胞 ,包装成重组腺病毒AdPF4。经鉴定后扩增、纯化、测病毒滴度及感染率。直接转导HUVEC检测表达蛋白活性。结果 :经鉴定此病毒DNA有PF4cDNA插入 ,病毒滴度可达 1× 10 1 0 pfu/ml,转导AdPF4病毒后的人脐静脉内皮细胞较对照含空载体病毒增殖减低 5 3 %。结论 :实验证实腺病毒介导的PF4cDNA体外具有抑制内皮细胞增殖活性。更多还原

【Abstract】 Objective:To construct the recombinant adenovirus of PF4cDNA by using the method of homogenouse recombination in bacteria,and to assess whether the proliferation of HUVEC transducted by recombinant adenovirus could be inhibited.Methods:The cDNA of PF4 was subcloned into the shuttle vector of pAdshuttle CMV,forming transfer vector of pAdshuttle/PF4.Then it was linearized with PmeI and cotransformed into BJ5183 cells with adenovirual geonomis plasmid of pAdEasy 1.The DNA of identified recombinant plasmid were digested with PacI and transfected to HEK293 cells to package adenovirus.The PCR technique was used to detect the target gene.The titres of recombinant adenovirus were measured by TCID50 method.The infection rate of recombinant adenovirus were assessed.Results:There were over 70% positive recombinant bacterial clones after cotransformation BJ5183 bacterial cells with pAdshuttle/PF4 and pAdEasy 1 by electroporation techneque.PCR indicated each the recombinant adenovirus contained the insert of PF4cDNA.The titres of recombinant adenovirus were 1×10 10 pfu/ml.The expressed PF4 significantly inhibits proliferation of endothelial cells in vitro.Conclusion:The recombinant adenovirus containing PF4cDNA could inhibite HUVEC proliferation.更多还原