基因变构人类白细胞介素-2克隆和原核表达-病原生物学专业论文.docxVIP

基因变扭厶娄臼绷胞众塞=2壶．隆垂!丝拄基丛基因变构人类白细胞介素一2克隆 基因变扭厶娄臼绷胞众塞=2壶．隆垂!丝拄基丛 基因变构人类白细胞介素一2克隆 和原核表达 青岛大学医学院二00一级病原生物学专业 硕士研究生 郑长青 导 师 王斌教授 摘 要 ①目的：通过定点诱变的方法，构建基因变构IL一2(”N—R)的重组克隆，并将其在 原核系统中进行高效表达，为研究基因变构IL-2(”N—R)的生物学活性奠定基础。 ②方法：从一名健康男子的扁桃体细胞中分离T细胞经PHA刺激活化后，提取细胞 的RNA为模板，逆转录构建cDNA文库，经套式PCR扩增出编码IL一2的基因，插入 T—A载体后获得编码天然IL一2的克隆，核苷酸测序证实成功后，自行设计含有突 变位点的引物，利用重组PCR技术，进行定点诱变构建基因变构IL一2(”N—R)的 重组克隆，DNA测序确认变构成功。将改构后的IL一2基因重组于原核pGEX-4T一2 质粒上，转化宿主菌E．coli．DH5 a中，经IPTG诱导后表达IL一2变构体，表达产 物经SDS电泳分析。③结果：获得了人类天然IL-2的编码基因克隆和基 因变构IL一2的编码基因克隆，并将变构IL一2(“N—R)在大肠杆菌中获得了高效表 达。④结论：IL一2基因的成功定点变构及其在原核生物中获得稳定高效的表达， 为进一步在真核细胞中表达以及研究制备低毒、高效的新型基因变构IL一2药物奠 定基础。 关键词 白细胞介素一2； 基因变构： 原核表达；DNA： 重组 注：本课题为山东省自然科学基金重点资助课题(E99C02) 基因变构人类白细胞介素一2克隆和原核表达The 基因变构人类白细胞介素一2克隆和原核表达 The Construction and Expression of a Site—specific Gene Mutant of The Human Interleukin一2 Gene in E．coli Postgraduate student：Zheng Changqing Tutor：WangBin Department of molecular Virology， Medical College of Qingdao University Abstract：0bjects：To construct expressive clones of a site—specific mutagenesis(“N—R1 of human interleukin一2(IL-2)gene with the aim of furthering the investigation of the new kind of hIL一2 genic drugs in clinical trial．Methods：The human Interleukin一2 cDNA containing full—length of encoding region was cloned by RT—PCR and confirmed by DNA sequencing．With recombinant PCR，We amplified the whole genes of IL-2 with the site— directed mutagenesis and confirmed by DNA sequencing again，then cloned to the procaryotic expressive vector pGEX·4T·2 and transformated to E．coli．DH5 Q．The IL-2 recombinant clone was induced with IPTG to express the allosteric IL一2 proteins and the products were identified by SDS·PAGEPAGE．Results：The mutated IL一2 gene clone was obtained successfully．the recombinant IL一2 proteins were efficiently expressed in E．coli DH5 Ct．Conclusion：Human IL·2 cDNA、vith the site—directed mutagenesis were expressed in E．coli，which has provided useful information for development of low—toxic， high·efficiency IL一2 recombinant drugs． KEY W