本文關(guān)鍵詞： sCTLA-4 sPD-L2-sCTLA-4 融合蛋白 免疫抑制　出處：《復(fù)旦大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：CTLA-4 (cytotoxic T lymphocyte associated antigen 4,細(xì)胞毒性T細(xì)胞相關(guān)抗原4)是一個(gè)表達(dá)于T細(xì)胞表面的蛋白分子,又稱(chēng)CD152,與CD28具有高度同源性,參與T細(xì)胞活化的負(fù)調(diào)控。PD-L2 (Programmed cell death 1 ligand 2)是B7家族的第5個(gè)成員,PD-L2和其受體PD-1結(jié)合后,可以下調(diào)TCR信號(hào),并最終抑制T細(xì)胞的活化。由于CTLA-4和PD-L2都能抑制病理性T細(xì)胞的活化,所以在器官移植和自身免疫病領(lǐng)域有著很好的前景。以分泌表達(dá)His-CTLA-4膜外可溶性片段的畢赤酵母菌株為材料,批量發(fā)酵表達(dá)得到純化蛋白,并在大鼠的類(lèi)風(fēng)濕性關(guān)節(jié)炎模型體內(nèi)驗(yàn)證了其功效。為了開(kāi)發(fā)治療器官移植排斥和自身免疫疾病的新潛在藥物,本實(shí)驗(yàn)室在先前研究的基礎(chǔ)上,重新構(gòu)建了不含His-tag的sCTLA-4畢赤酵母表達(dá)株,并通過(guò)多種方法對(duì)表達(dá)水平進(jìn)行優(yōu)化,但由于表達(dá)量仍然不理想并且不帶標(biāo)簽,后續(xù)純化步驟很難進(jìn)行。因此我們重新構(gòu)建了含His-tag和凝血酶識(shí)別切割位點(diǎn)的sCTLA-4表達(dá)載體pET-sCTLA-4,結(jié)果成功的表達(dá)和純化了sCTLA-4蛋白,用凝血酶切除His-tag后,進(jìn)一步的功能實(shí)驗(yàn)表明,在sCTLA-4終濃度為10ng/μl時(shí),對(duì)小鼠雙向混合淋巴細(xì)胞反應(yīng)和ConA轉(zhuǎn)化實(shí)驗(yàn)的抑制率分別達(dá)到69%和50%,具有顯著的體外細(xì)胞免疫抑制功能。另外,我們通過(guò)生物信息學(xué)原理及生物大分子結(jié)構(gòu)與功能關(guān)系和免疫學(xué)相關(guān)知識(shí),用基因工程的方法設(shè)計(jì)了一個(gè)新的雙功能細(xì)胞分子——sCTLA-4-sPD-L2融合蛋白,它具有sCTLA-4和sPD-L2分子的雙重功能。通過(guò)小鼠體外雙向混合淋巴細(xì)胞反應(yīng)、淋巴細(xì)胞轉(zhuǎn)化實(shí)驗(yàn)表明sCTLA-4-sPD-L2融合蛋白能顯著抑制小鼠淋巴細(xì)胞的活化,初步證明了其免疫負(fù)調(diào)控功能,為開(kāi)發(fā)新型免疫抑制藥物奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：Q789;R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王麗莎;朱乃碩;;PD-L2/CTLA-4融合蛋白的表達(dá),純化及初步功能鑒定[J];復(fù)旦學(xué)報(bào)(自然科學(xué)版);2014年04期

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本文編號(hào)：1515123