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

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.