免疫负调控分子sCTLA-4和sPD-L2-sCTLA-4融合蛋白的基因工程表达和初步功能研究

发布时间：2018-02-16 08:48

本文关键词： sCTLA-4 sPD-L2-sCTLA-4 融合蛋白免疫抑制　出处：《复旦大学》2012年硕士论文　论文类型：学位论文

【摘要】：CTLA-4 (cytotoxic T lymphocyte associated antigen 4,细胞毒性T细胞相关抗原4)是一个表达于T细胞表面的蛋白分子,又称CD152,与CD28具有高度同源性,参与T细胞活化的负调控。PD-L2 (Programmed cell death 1 ligand 2)是B7家族的第5个成员,PD-L2和其受体PD-1结合后,可以下调TCR信号,并最终抑制T细胞的活化。由于CTLA-4和PD-L2都能抑制病理性T细胞的活化,所以在器官移植和自身免疫病领域有着很好的前景。以分泌表达His-CTLA-4膜外可溶性片段的毕赤酵母菌株为材料,批量发酵表达得到纯化蛋白,并在大鼠的类风湿性关节炎模型体内验证了其功效。为了开发治疗器官移植排斥和自身免疫疾病的新潜在药物,本实验室在先前研究的基础上,重新构建了不含His-tag的sCTLA-4毕赤酵母表达株,并通过多种方法对表达水平进行优化,但由于表达量仍然不理想并且不带标签,后续纯化步骤很难进行。因此我们重新构建了含His-tag和凝血酶识别切割位点的sCTLA-4表达载体pET-sCTLA-4,结果成功的表达和纯化了sCTLA-4蛋白,用凝血酶切除His-tag后,进一步的功能实验表明,在sCTLA-4终浓度为10ng/μl时,对小鼠双向混合淋巴细胞反应和ConA转化实验的抑制率分别达到69%和50%,具有显著的体外细胞免疫抑制功能。另外,我们通过生物信息学原理及生物大分子结构与功能关系和免疫学相关知识,用基因工程的方法设计了一个新的双功能细胞分子——sCTLA-4-sPD-L2融合蛋白,它具有sCTLA-4和sPD-L2分子的双重功能。通过小鼠体外双向混合淋巴细胞反应、淋巴细胞转化实验表明sCTLA-4-sPD-L2融合蛋白能显著抑制小鼠淋巴细胞的活化,初步证明了其免疫负调控功能,为开发新型免疫抑制药物奠定了基础。
[Abstract]:CTLA-4 cytotoxic T lymphocyte associated antigen 4 (cytotoxic T cell associated antigen 4) is a protein molecule expressed on the surface of T cells, also known as CD152, and has high homology with CD28. PD-L2 Programmed cell death 1 ligand 2), a fifth member of the B7 family, can down-regulate TCR signal and ultimately inhibit the activation of T cells after binding to its receptor PD-1. Both CTLA-4 and PD-L2 can inhibit the activation of pathological T cells. Therefore, there is a good prospect in the field of organ transplantation and autoimmune disease. Using Pichia pastoris strain secreting soluble fragments of His-CTLA-4 extracellular membrane as material, the purified protein was obtained by batch fermentation. To develop new potential drugs for the treatment of organ transplant rejection and autoimmune diseases, our lab has built on previous studies to test its efficacy in rats with rheumatoid arthritis. A sCTLA-4 Pichia pastoris expression strain without His-tag was constructed, and the expression level was optimized by a variety of methods. However, the expression level is still not ideal and untagged. Therefore, we reconstructed the sCTLA-4 expression vector pET-sCTLA-4 containing His-tag and thrombin recognition site, and successfully expressed and purified the sCTLA-4 protein. After His-tag was removed by thrombin, further functional experiments showed that pET-sCTLA-4 was used to express and purify pET-sCTLA-4. When the final concentration of sCTLA-4 was 10 ng / 渭 l, the inhibition rates of bidirectional mixed lymphocyte reaction and ConA transformation were 69% and 50, respectively. We have designed a novel bifunctional cellular molecule, sCTLA-4-sPD-L2 fusion protein, based on the bioinformatics principle, the relationship between the structure and function of biological macromolecules and immunological knowledge, and by genetic engineering, we designed a novel bifunctional cellular molecule, sCTLA-4-sPD-L2 fusion protein. It has the dual function of sCTLA-4 and sPD-L2 molecules. The lymphocyte transformation test shows that sCTLA-4-sPD-L2 fusion protein can significantly inhibit the activation of mouse lymphocytes, which preliminarily proves its negative immune regulation function. It lays a foundation for the development of new immunosuppressive drugs.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：Q789;R392

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