人IL-15在K562细胞中的表达研究

发布时间：2018-04-17 10:20

本文选题：白细胞介素15 + 克隆　；参考：《吉林大学》2014年硕士论文

【摘要】：白细胞介素15（Interleukin-15,IL-15）是一种多能的细胞因子，能够促进淋巴细胞和NK细胞的增殖且对免疫系统中各种免疫细胞都具有重要的调节作用。IL-15表现出与IL-2在抗肿瘤、免疫调节等方面相似的生物学作用而备受关注。本实验利用RT-PCR技术从人外周血单个核细胞中克隆了人IL-15cDNA部分序列，并以其为模板分别扩增出IL-15编码序列（coding sequence，CDS）以及引入CD8α跨膜肽的IL-15编码序列，利用基因组技术构建分泌型人IL-15克隆载体pMD T-IL-15、表达载体pVITRO2-IL-15以及膜结合型IL-15克隆载体pMD T-mbIL-15、表达载体pVITRO2-mbIL-15。分别将表达载体转染K562细胞，检测分泌型IL-15在K562细胞中表达情况，并通过细胞流式技术鉴定，以期获得能稳定表达的膜结合IL-15的K562细胞株。对进一步研究其在免疫系统中的重要作用，并为其在肿瘤基因治疗、免疫治疗等奠定了基础。结果表明，本实验成功克隆了人IL-15CDS序列，，在此基础上正确构建了重组人IL-15分泌型真核表达载体pVITRO2-IL-15和重组人IL-15跨模型真核表达载体pVITRO2-mbIL-15。最后通过将重组真核表达载体分别转染K562细胞，检测了分泌型IL-15在K5625细胞中的表达，并获得稳定表达膜结合IL-15蛋白的重组K562细胞株。
[Abstract]:Interleukin 15 Interleukin-15 (IL-15) is a multipotent cytokine that promotes the proliferation of lymphocytes and NK cells and plays an important role in regulating all immune cells in the immune system. IL-15 is associated with IL-2 in anti-tumor activity.Immunomodulation and other similar biological functions have attracted much attention.In this study, we cloned human IL-15cDNA partial sequence from human peripheral blood mononuclear cells by RT-PCR technique, and used it as template to amplify the coding sequence of IL-15 coding sequence sequence sequence and the IL-15 coding sequence that introduced CD8 伪 transmembrane peptide.The secretory human IL-15 clone vector pMD T-IL-15, the expression vector pVITRO2-IL-15 and the membrane-binding IL-15 clone vector pMD T-mbIL-15 were constructed by genomic technique. The expression vector pVITRO2-mbIL-15 was constructed.The expression vectors were transfected into K562 cells, and the expression of secretory IL-15 in K562 cells was detected. The expression of secretory IL-15 in K562 cells was identified by flow cytometry.It can be used to further study its important role in the immune system and lay a foundation for its application in tumor gene therapy and immunotherapy.The results showed that the human IL-15CDS sequence was cloned successfully, and the recombinant human IL-15 secreted eukaryotic expression vector pVITRO2-IL-15 and the recombinant human IL-15 transmodel eukaryotic expression vector pVITRO2-mbIL-15 were constructed.Finally, the recombinant eukaryotic expression vector was transfected into K562 cells, and the expression of secretory IL-15 in K5625 cells was detected, and a recombinant K562 cell line with stable expression of membrane binding IL-15 protein was obtained.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

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