南京医科大学学报（自然科学版）2011，Vol.31Issue(3)：283-288,6.
含KSHV vIL-6基因重组分泌型表达载体的构建及其分泌蛋白对内皮细胞增殖和血管生成能力影响的初探
Construction of the recombinant secretory expression plasmid containing KSHV vlL-6 gene and effect of the secretory protein on proliferation and angiogenesis of vascular endothelial cells
摘要
Abstract
Objective:To construct the recombinant secretory plasmid containing vlL-6 gene of KSHV,detect its protein expression in EA.hy926 cells,and observe the effect of the secretory protein on proliferation and angiogenesis of vascular endothelial cells.Methods:A pair of PCR primers for vIL-6 gene including HindⅢ and Xho Ⅰ restriction sites was designed according to the sequence of pvIL-6 F. vIL-6 gene was amplified by PCR, taking pvIL-6 F as template. Then purified vIL-6 gene fragments were digested and cloned into pSecTag2B vector to generate recombinant secretory expression plasmid named as pSecTag2B-vIL-6. pSecTag2B-vIL-6 was transfected into EA.hy926 cells. The expression of vIL-6 protein in EA.hy926 cells was detected by Western blot, and supernatant of 293T cells transfected by the recombinant secretory plasmid was collected, then the effect of vIL-6 on proliferation and angiogenesis of vascular endothelial cells by autocrine and paracrine was detected by cell proliferation assay and microtube formation assay. Results:The recombinant secretory plasmid carrying KSHV vIL-6 gene was constructed successfully. After transfected with the above plasmid, the exact band of vIL-6 was detectable by Western blot. EA.hy926 ceils treated either with supernatants collected from 293T cells transfected by recombinant secretory plasmid or with transient transfection of the recombinant secretory plasmid, could perform an enhancement in proliferation and angiogenesis. Conclusion:The recombinant secretory plasmid pSecTag2B-vIL-6 was constructed successfully, and vIL-6 protein could be correctly expressed in EA.hy926 cells. Importantly, vIL-6 protein could promote the proliferation and angiogenesis of vascular endothelial cells through autocrine and paracfine pathway.关键词
KSHV/vIL-6/分泌型/细胞增殖/血管生成Key words
KSHV/vIL-6/eukaryotic secretory expression/cell proliferafion/angiogenesis分类
医药卫生引用本文复制引用
朱小飞,秦娣,卢春..含KSHV vIL-6基因重组分泌型表达载体的构建及其分泌蛋白对内皮细胞增殖和血管生成能力影响的初探[J].南京医科大学学报（自然科学版）,2011,31(3):283-288,6.基金项目
国家自然科学基金(30900064) （30900064）
教育部高等学校博士点专项科研基金新教师基金课题(20093234120004) （20093234120004）
江苏省高校自然科学基金(09KJB310007)资助 （09KJB310007）
