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目的:构建鸡白细胞介素2(IL-2)真核重组表达质粒,体外鉴定其能否表达。方法:将鸡IL-2cDNA(RT-PCR法获得),克隆入真核高效表达质粒pcDNA3.1(+)中,构建pcDNA-IL-2重组表达质粒。PCR和双酶切的方法鉴定克隆的正确性。然后构建pcDNA IL GFP重组表达质粒(即GFP基因与IL2的一段上游基因融合表达),PCR方法鉴定克隆的正确性。脂质体法转染COS1细胞,荧光显微镜下观察荧光。结果:正确构建了真核重组表达质粒pcDNA-IL2和pcDNA-IL-GFP。荧光显微镜下可观察到很强的绿色荧光。结论:鸡IL-2克隆和表达成功。为下一步用重组质粒pcDNA-IL2免疫BALB/c小鼠,研制鸡IL-2单克隆抗体奠定了基础。
Objective: To construct eukaryotic recombinant plasmid of interleukin 2 (IL-2) and identify its expression in vitro. Methods: Chicken IL-2 cDNA (obtained by RT-PCR) was cloned into eukaryotic expression plasmid pcDNA3.1 (+) to construct recombinant plasmid pcDNA-IL-2. PCR and double digestion method to identify the correctness of the clones. Then construct recombinant plasmid pcDNA IL GFP (ie GFP gene and IL2 an upstream gene fusion expression), PCR method to identify the correctness of the clone. COS1 cells were transfected by lipofectamine and the fluorescence was observed under fluorescence microscope. Results: Eukaryotic recombinant plasmids pcDNA-IL2 and pcDNA-IL-GFP were constructed correctly. Fluorescent microscope can be observed under the strong green fluorescence. Conclusion: The chicken IL-2 was cloned and expressed successfully. In order to further immunize BALB / c mice with the recombinant plasmid pcDNA-IL2, chicken IL-2 monoclonal antibody was developed.