IL-1Ra和IL-10真核表达质粒载体的构建及其表达检测  

Construction and Identification of Eukaryotic Expression Plasmid Encoding Human IL-1Ra and IL-10

作　　者：傅欣[1] 郁时兵[1] 张晓玲[1] 张继英[1] 毛泽斌[2] 于长隆[1] 

机构地区：[1]北京大学第三医院运动医学研究所,北京100083 [2]北京大学医学部生物化学与分子生物学系

出　　处：《中国运动医学杂志》2006年第1期54-56,100,共4页Chinese Journal of Sports Medicine

基　　金：国家体育总局科技攻关重点项目(No.04014)资助

摘　　要：目的:构建人IL-1Ra和人IL-10真核表达质粒载体并检测其表达。方法:用双酶切方法切取PCDI-IL-1Ra和PCDI-IL-10质粒中包含人IL-1Ra和人IL-10 CDS全长序列的cD-NA片段,并分别连接到真核表达质粒pcDNA3.1上,然后用壳聚糖转染上述质粒到原代软骨细胞,RT-PCR检测其mRNA水平的表达。结果:成功将人IL-1Ra和IL-10 CDS全长序列的cDNA片段克隆到真核表达载体,mRNA水平检测到目的基因的表达明显提高。结论:真核表达质粒可以用于外源基因的原代软骨细胞导入和表达,为进一步的基因治疗研究提供依据。Objective To construct efficient expression plasmid encoding human IL- 1Ra and IL- 10, and provide an important tool for IL- IRa and IL- 10 gene therapy. Methods Plasmid PCDI- IL- 1Ra was treated with endonucleases EcoR Ⅰ and Hind Ⅲ , the the product was ligated with digested vector PcDNA3.1 （ - ） by EcoR Ⅰ and Hind Ⅲ . Plasmid PCDI - IL - 10 was similarly treated with endonucleases BamH Ⅰ and Kpn Ⅰ , and then the product was ligated with digested vector PcDNA3.1 （ ＋ ） by BamH Ⅰ and Kpn Ⅰ . The resultant recombinant plasmids were identified with appropriate endonucleases and sequencing. The transfection efficiency of the constructed plasmids to primary chondrocytes were determined by RT - PCR. Results The human IL- 1Ra and IL- 10 eDNA were certified to be inserted into PcDNA3.1 and its expression in primary chondrocytes improved significantly. Conclusion Cytokines gene can be transfected to primary cells through eukaryotic expression plasmid.

关 键 词：白介素1受体拮抗剂 白介素10 真核质粒 表达 

分 类 号：R684.3[医药卫生—骨科学]