【作者】 宋世斌；

【导师】 宗瑞谦； 景志忠；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 白细胞介素-18是新近发现的一种细胞因子,它具有诱导T细胞和NK细胞产生IFN-γ,诱导GM-CSF的分泌,增强T细胞和NK细胞的细胞毒作用等免疫调节功能;能发挥显著的免疫佐剂作用;在抗肿瘤、病原微生物感染及抗超敏反应等方面有着潜在的应用前景。本论文取得的主要研究结果如下:采用PCR技术从pGEM-IL-18重组质粒中扩增出IL-18基因并构建真核融合表达载体pPIC9K-IL-18,电激法转化入毕赤酵母GS115,采用G418抗性梯度法筛选得到多拷贝重组菌株,甲醇诱导表达,应用SDS-PAGE分析重组蛋白的表达情况,并将表达蛋白用凝胶层析柱纯化后,用MTT法检测其生物学活性。实验结果表明重组的GS115酵母菌株可表达分泌pIL-18,其表达在72h时达高峰,分泌量可达160mg/ L ,纯化的重组pIL-18蛋白具有显著的促进淋巴细胞增殖的活性,说明本试验在国内首次在毕赤酵母中成功表达了具有生物学活性的pIL-18。从发酵时间、不同诱导型、pH值、诱导剂量和接种量等方面对重组基因工程菌的发酵条件在摇床水平上进行了优化,进一步探索了酵母菌表达猪IL-18的发酵工艺。初步建立了一套较完善的在毕赤酵母中表达猪IL-18基因的生产工艺条件,为在发酵罐水平上大规模表达猪IL-18提供了依据。结果表明,摇瓶发酵时,诱导最佳时间为72h,甲醇最适浓度为20g/L,发酵pH范围为6.0～8.0,最适接种量1∶1;与单纯甲醇诱导相比,甘油/甲醇诱导后的表达量明显提高。本研究为猪IL-18基因工程新制剂的生产和应用奠定了基础。更多还原

【Abstract】 IL-18 was lately discovered, involved in induction of production of IFN-γand GM-CSF secreted by T and NK cell and in enforcement of cytotoxic effect of T and NK cell. It has promising prospects application in the fields of anti-tumor, infection of pathogenic microbes as well as hypersensitiveness. In this study, we have obtained the following results:The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR, then the fusion expression vector pPIC9K-IL-18 was constructed by inserting IL-18 fragment, and transformed to GS115 by electroporation. Multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol, and SDS-PAGE was used to analyze the product. The bioactivity of IL-18 purified by Sephadex G-100, was measured by MTT assay. Experiment results showed the fusion protein of IL-18 secreted by GS115 and reached the expression peak of 160mg/L at 72h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in pichia pastoris.Except the internal characters of heterogenous gene sequence itself, expression conditions also plays important roles in the expression yield. Different culturable time, inducible patterns, pH valves and the amount of inducible reagent were compared on effect of expression in this paper respectively. Experimental results revealed that the fermentation period induced with methanol was 72 h. The optimal concentration of methanol was 20g/L, pH range was 6.0–8.0 and the inoculation amount seed was 1: 1 in shake-flask fermentation. Compared with those induced only with methanol, pattern of inhibition/induction could improve the expression level significantly.This study provides bases on development and application of new type reagent porcine IL-18.更多还原