文章摘要
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,2011,(3):283~288
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 vIL-6 gene and effect of the secretory protein on proliferation and angiogenesis of vascular endothelial cells
投稿时间:2010-09-30  
DOI:10.7655
中文关键词: KSHV  vIL-6  分泌型  细胞增殖  血管生成
英文关键词: KSHV  vIL-6  eukaryotic secretory expression  cell proliferation  angiogenesis
基金项目:国家自然科学基金(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);江苏省高校自然科学基金(09KJB310007)资助
作者单位
朱小飞 南京医科大学微生物学与免疫学系,江苏 南京 210029 
秦 娣 南京医科大学微生物学与免疫学系,江苏 南京 210029 
卢 春 南京医科大学微生物学与免疫学系,江苏 南京 210029 
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中文摘要:
      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?
英文摘要:
      Objective:To construct the recombinant secretory plasmid containing vIL-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 XhoⅠ 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 cells 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 paracrine pathway.
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