免疫毒素IL-3-PE38KDEL原核表达载体的构建

发布时间：2018-05-25 23:53

本文选题：重组毒素 + 人白介素3　；参考：《重庆医科大学》2008年硕士论文

【摘要】： 目的:构建人白细胞介素(IL-3)与假单胞菌外毒素片段(PE38KDEL)融合基因的原核表达载体,为进一步表达具有杀伤作用的融合蛋白奠定基础。方法: 1)提取人外周血总RNA,经RT-PCR扩增出IL-3基因,并用二次PCR方法引入柔性连接肽linker片段,PCR产物插入载体PQE30中,转化克隆进行酶切鉴定及DNA测序肯定克隆结果。 2)以PGEX-4T-1PE38KDEL质粒为模板,PCR扩增PE38KDEL片段,PCR产物经双酶切后插入到PQE30-IL-3质粒中构建PQE30-IL-3-PE38KDEL,连接产物转化克隆经酶切鉴定及DAN测序鉴定。结果:本研究成功构建了原核表达质粒PQE30-IL-3-PE38KDEL.经酶切鉴定与预期结果吻合,核酸序列测定显示插入的IL-3基因及PE38KDEL基因与Genebank报道的序列完全一致。
[Abstract]:Aim: to construct the prokaryotic expression vector of the fusion gene of human interleukin 3 (IL 3) and pseudomonas exotoxin fragment PE38 KDEL, and to lay a foundation for the further expression of the fusion protein with killing effect. Methods: 1) Human peripheral blood total RNAs were extracted, and IL-3 gene was amplified by RT-PCR, and then inserted into the vector PQE30 by the second PCR method. The transformed clones were identified by restriction endonuclease digestion and the results were confirmed by DNA sequencing. 2) PGEX-4T-1PE38KDEL plasmid was used as template to amplify the PE38KDEL fragment. The product was digested by double enzyme and inserted into PQE30-IL-3 plasmid to construct PQE30-IL-3-PE38KDEL. The ligated product was identified by restriction endonuclease digestion and DAN sequencing. Results: the prokaryotic expression plasmid PQE30-IL-3-PE38 KDEL was successfully constructed. The nucleic acid sequence analysis showed that the inserted IL-3 gene and PE38KDEL gene were identical with the sequence reported by Genebank.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1

【参考文献】