| KiSS-1基因重组真核质粒的构建、鉴定及其表达 |
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| 引用本文： | 徐玫芳,臧盛兵,黄爱民,刘景丰,高凌云,高美钦.KiSS-1基因重组真核质粒的构建、鉴定及其表达[J].福建医科大学学报,2009,43(3):193-197. |
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| 作者姓名： | 徐玫芳 臧盛兵 黄爱民 刘景丰 高凌云 高美钦 |
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| 作者单位： | 1. 福建医科大学基础医学院,病理学系,肿瘤研究室,福州350004
2. 福建医科大学附属第一医院,肝病中心,福州350005 |
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| 基金项目： | 福建省教育厅科研项目;福建医科大学教授学术发展基金 |
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| 摘 要： | 目的构建KiSS-1基因重组真核质粒，将其导入人高转移肝癌MHCC97-H细胞中，获得瞬时表达的细胞株，为进一步研究KISS-1基因对人肝癌细胞侵袭转移的影响奠定基础。方法Trizol试剂法提取新鲜胎盘组织总RNA，经RT—PCR扩增目的片段，PCR产物及真核表达载体分别双酶切后进行连接．并转化人大肠杆菌Ecoli．Dh5α扩增以获得重组载体，采用脂质体介导转染技术将高纯度的KiSS-1质粒转染到人高转移肝癌细胞株中，用RT—PCR和Westernblot技术加以鉴定。结果RT—PCR获得预期大小的特异性DNA片段，经PCR、双酶切鉴定及测序，证实已将KISS-1基因eDNA片段正确插入真核表达载体中；RT-PCR、免疫细胞化学和Westernblot证实KiSS-1基因已转染入MHCC97-H细胞中。结论成功获得pcDNA3．1／HisC／KiSS-1重组质粒和瞬时表达KiSS-1基因的肝癌细胞，为后续建立稳定转染的KiSS-1高表达阳性细胞克隆奠定基础，为体内外实验研究KiSS-1对人肝癌细胞增殖、侵袭和转移能力的影响提供前提条件。
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| 关 键 词： | 肿瘤抑制蛋白类 遗传载体 转染 肝肿瘤 肿瘤侵润 真核细胞核 质粒 |
Construction,Identification and Expression of Recombinant Eukaryotic Expression Vector of Human KiSS-1 Gene |
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| XU Meifang,ZANG Shengbing,HUANG Aimin,LIU Jingfeng,GAO Lingyun,GAO Meiqin.Construction,Identification and Expression of Recombinant Eukaryotic Expression Vector of Human KiSS-1 Gene[J].Journal of Fujian Medical University,2009,43(3):193-197. |
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| Authors: | XU Meifang ZANG Shengbing HUANG Aimin LIU Jingfeng GAO Lingyun GAO Meiqin |
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| Institution: | XU Meifang1,ZANG Shengbing1,HUANG Aimin1,LIU Jingfeng2,GAO Lingyun1,GAO Meiqin1 1.Department of Pathology,School of Basic Medical Sciences,Fujian Medical University,Institute of Oncology,Fuzhou 350004,China,2.Hepatopathy Center,The First Affiliated Hospital,Fuzhou 350005 |
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| Abstract: | Objective To construct a recombinant eukaryotic expression vector containing the functional region of human KiSS-1 gene and then to transfect this vector into human hepatocellular carcinoma cell line MHCC97-H with high metastasis potential.Transient-expression cell lines are to be obtained so as to establish a foundation for a further study on the impact of KiSS-1 on hepatoma carcinoma cell invasion and metastasis.Methods Total RNA was extracted by using Trizol one-step method from fresh placenta tissues.cD... |
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| Keywords: | tumor suppressor protein genetic vectors transfection liver neoplasms neoplasm invasiveness eukaryotic cells plasmids |
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