基因重组真菌免疫调节蛋白的筛选与功能检测

发布时间：2018-06-14 12:54

本文选题：真菌免疫调节蛋白 + 菌落杂交筛选　；参考：《上海交通大学》2012年硕士论文

【摘要】：真菌免疫调节蛋白(fugal immunomodulatory proteins,FIPs)是从高等担子菌中分离出来的一类具有免疫调节活性的小分子蛋白质,目前已经从多种高等担子菌中分离得到天然蛋白质以及克隆得到fip基因序列,真菌免疫调节蛋白具有广泛的免疫调节特性,如促进淋巴细胞的增殖,调控细胞周期,诱发细胞凋亡,影响细胞因子的表达,活化细胞粘附分子,抗过敏反应等等,具有巨大的蛋白质药物研究开发潜力。本实验将fip基因根据其来源分成两组:属内组(fip-glu和fip-gsi)和属间组(fip-glu、fip-fve和fip-vvo),利用DNA shuffling技术对这两组基因在分子水平上进行重组,构建得到改组文库,利用斑点杂交技术和特异性抗体对改组文库共筛选426株属内重组菌株和412株属间组菌株。经过序列分析和蛋白诱导表达分析,属内组和属间组基因重组蛋白完全符合改组模式,检测重组蛋白的相对表达产量并筛选得到相对表达产量明显提高的菌株;纯化蛋白并进行血细胞凝集实验以及小鼠脾细胞因子诱导表达分析,实验证明当属内组FIP和属间组FIP的浓度不低于0.20μg ml-1和1.56μg ml-1时,能够凝集小鼠血细胞;重组FIP能够诱导小鼠脾细胞因子interleukin(IL)-2, IL-3, IL-4, interferon(IFN)-γ, IL-2 receptor(IL-2R)和lymphotoxin(LT)基因的转录水平。该项研究使利用基因工程和发酵技术进行目的产物的产业化生产将逐渐成为可能,同时有助于探索该蛋白质家族编码基因的表达调控关系。
[Abstract]:Fungal immunomodulatory protein (immunomodulatory proteinsus) is a kind of small molecular protein with immunomodulatory activity isolated from higher basidiomycetes. At present, natural proteins have been isolated from a variety of higher basidiomycetes and fip gene sequences have been cloned. Fungal immunoregulatory proteins have a wide range of immunomodulatory properties, such as promoting the proliferation of lymphocytes, regulating cell cycle, inducing apoptosis, affecting the expression of cytokines, activating cell adhesion molecules, anti-allergic reactions and so on. It has great potential in protein drug research and development. In this study, fip genes were divided into two groups according to their origin: intra group fip-glu and fip-gsi. and intergeneric group, fip-glufifip-fve and fip-vvov. The two groups of genes were recombined at molecular level by shuffling technique, and the recombinant library was constructed. A total of 426 intrageneric recombinant strains and 412 intergeneric strains were screened by dot blot and specific antibody library. After sequence analysis and protein induced expression analysis, the recombinant proteins of intra and intergeneric groups were completely in accordance with the shuffling model. The relative expression yield of recombinant proteins was detected and the strains with higher relative expression yield were screened. The purified protein was purified by hemagglutination assay and the expression of spleen cytokines in mice was analyzed. The results showed that when the concentrations of intra and intergeneric FIP were not less than 0.20 渭 g ml-1 and 1.56 渭 g ml-1, the blood cells of mice could be agglutinated. Recombinant FIP can induce the transcription levels of interleukin-2, IL-3, IL-4, IFN- 纬, IL-2 receptor IL-2R) and lymphotoxin LTs genes in mice. This study will make it possible to use genetic engineering and fermentation technology to produce the target product, and it will be helpful to explore the expression and regulation of the protein family coding gene.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

【引证文献】