文章摘要
程晓东,薛 敏,郝婷婷,秦 娣,卢 春.卡波氏肉瘤病毒K1蛋白在血管内皮细胞中的表达及其功能初探[J].南京医科大学学报,2013,(5):586~592
卡波氏肉瘤病毒K1蛋白在血管内皮细胞中的表达及其功能初探
Expression of KSHV K1 protein in vascular endothelial cells and preliminary study on its function
投稿时间:2012-10-30  
DOI:10.7655/NYDXBNS20130505
中文关键词: KSHV K1蛋白  增殖  迁移
英文关键词: KSHV K1 protein  proliferation  migration
基金项目:教育部高等学校博士点专项科研基金新教师基金课题 (20093234120004)
作者单位
程晓东 南京医科大学微生物学与免疫学系,江苏 南京 210029 
薛 敏 南京医科大学微生物学与免疫学系,江苏 南京 210029 
郝婷婷 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:获得稳定表达卡波氏肉瘤病毒(Kaposi’s sarcoma-associated herpesvirus,KSHV)K1蛋白的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),探究K1蛋白对HUVECs增殖和迁移能力的影响。方法:从本实验室已构建好的含KSHV K1基因的重组真核表达质粒pCI-neo-K1中扩增出K1基因,克隆入慢病毒载体pHAGE-CMV-MCS-IZs-Green中构建重组质粒pHAGE-K1。利用脂质体将pHAGE-K1与包装质粒psPAX2和包膜质粒pMD2.G共转染293T细胞,收集培养上清经0.45 μm滤膜过滤即获得慢病毒悬液。病毒感染HUVECs,Western blot检测K1蛋白的表达。通过绿色荧光蛋白进行流式分选?Western blot验证获得稳定表达K1蛋白的HUVECs。通过细胞增殖实验和细胞划痕实验检测K1蛋白对HUVECs增殖和迁移能力的影响。结果:核酸序列测定证实,克隆的K1基因全长906 bp,与GenBank中登记的K1基因100%同源。Western blot结果显示,含K1基因的重组慢病毒感染的HUVECs在约46 000处可观察到一特异性条带,与预期的K1蛋白大小一致。流式分选获得稳定表达K1蛋白的HUVECs,其增殖能力与对照组相比显著增强(P < 0.01),细胞迁移能力亦明显增加(P < 0.05)。结论:成功包装了含KSHV K1基因的重组慢病毒。KSHV K1蛋白能够促进HUVECs增殖和迁移。
英文摘要:
      Objective:To obtain human umbilical vein endothelial cells (HUVECs) that stably expressing KSHV K1 protein and explore the effect of K1 protein on the proliferation and migration ability of HUVECs. Methods:The constructed fragment of K1 gene from expression plasmid pCI-neo-K1 was cloned into the lentiviral vector pHAGE-CMV-MCS-IZs-Green. We co-transfected the recombinant plasmid pHAGE-K1,the packaging plasmid psPAX2 and the envelope plasmid pMD2.G into 293T cells. Culture media were harvested and filtered through a 0.45 μm filter to remove the cells. The expression of K1 protein in recombinant lentivirus-infected HUVECs was detected by Western blot assay. Next,HUVECs stably expressing KSHV K1 protein were obtained by flow cytometry assay (FCM) screening and verified the expression of K1 protein by Western blot assay again. Finally,the effect of K1 protein on the proliferation and migration ability of HUVECs was detected by CCK-8 assay and wound-healing assay. Results:Nucleic acid sequencing confirmed that cloned K1 gene was 906 bp,wihch was 100% homologous with K1 gene registered in GenBank. The exact band of K1 protein in recombinant lentivirus-infected HUVECs was detectable by Western blot assay. The results of CCK-8 and wound-healing assay showed that the proliferation and migration ability of HUVEC stably expressing KSHV K1 protein was significantly increased than the corresponding control(P < 0.01 and P < 0.05,respectively). Conclusion:The recombinant lentivirus carrying KSHV K1 gene was packaged successfully,and K1 protein could promote the proliferation and migration of HUVECs.
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