论文部分内容阅读
目的:构建含卡波济肉瘤相关疱疹病毒(Kaposi’s sarcoma-assoliated herpesvirus,KSHV)致瘤基因vIL-6的重组分泌型质粒,将其导入内皮细胞系EA.hy926中进行表达,并检测vIL-6对内皮细胞增殖和血管生成作用的影响。方法:根据本实验室先前构建的pvIL-6 F表达载体中的核酸序列设计PCR引物,在其5′端及3′端分别引入HindⅢ和XhoⅠ酶切位点,PCR扩增vIL-6基因,经双酶切纯化后将其克隆入分泌型真核表达载体pSecTag2B中。重组质粒经核酸测序鉴定后转染EA.hy926细胞,以Western blot检测vIL-6基因的表达情况;另外收集重组分泌型质粒转染293T细胞后的上清,通过细胞增殖实验和微管形成实验分别检测vIL-6通过自分泌或旁分泌方式对内皮细胞增殖和血管生成作用的影响。结果:限制性内切酶鉴定和基因测序证实成功构建了含KSHV vIL-6基因的重组分泌型质粒pSecTag2B-vIL-6,此质粒转染EA.hy926细胞后,Western blot能够在相应位置检测到目的蛋白vIL-6的条带。通过将pSecTag2B-vIL-6瞬时转染EA.hy926细胞或者收集pSecTag2B-vIL-6转染293T细胞后的上清作用于EA.hy926细胞,均观察到细胞增殖和血管生成能力的明显增强。结论:成功构建了重组分泌型质粒pSecTag2B-vIL-6;目的蛋白可在EA.hy926细胞中表达,且vIL-6能够通过自分泌和旁分泌方式促进内皮细胞的增殖和血管生成。
OBJECTIVE: To construct a recombinant secretory plasmid containing the vIL-6 gene of Kaposi’s sarcoma -assoliated herpesvirus (KSHV), which was then introduced into endothelial cell line EA.hy926 for expression. The expression of vIL-6 Effects on Endothelial Cell Proliferation and Angiogenesis. Methods: PCR primers were designed based on the nucleic acid sequence of pvIL-6 F expression vector previously constructed in our laboratory. Hind Ⅲ and Xho Ⅰ restriction sites were introduced into the 5 ’and 3’ ends respectively. The vIL-6 gene was amplified by PCR. After purification by double digestion, it was cloned into secretory eukaryotic expression vector pSecTag2B. The recombinant plasmids were identified by nucleic acid sequencing and transfected into EA.hy926 cells. The expression of vIL-6 gene was detected by Western blot. The supernatant of 293T cells transfected with the recombinant secreted plasmids was also collected. Through cell proliferation assay and microtubule formation assay The effects of vIL-6 on endothelial cell proliferation and angiogenesis were examined by autocrine or paracrine methods respectively. Results: The restriction endonuclease and gene sequencing confirmed that the recombinant secretory plasmid pSecTag2B-vIL-6 containing KSHV vIL-6 gene was successfully constructed. After transfected into EA.hy926 cells, Western blot can be detected at the corresponding position The target protein vIL-6 band. The proliferation and angiogenesis ability of EA.hy926 cells were observed by transient transfection of pSecTag2B-vIL-6 into EA.hy926 cells or the supernatant of 293T cells transfected with pSecTag2B-vIL-6. CONCLUSION: The recombinant secretory plasmid pSecTag2B-vIL-6 has been successfully constructed. The target protein can be expressed in EA.hy926 cells and vIL-6 can promote endothelial cell proliferation and angiogenesis through autocrine and paracrine processes.