摘要
背景:热休克蛋白70的肿瘤免疫作用近年来受到国内外学者的广泛关注,然而目前常用的诱导内源性热休克蛋白70表达的方法,如热应激、缺血预处理等均存在一些弊端。目的:拟构建携带并正确表达外源性人热休克蛋白70基因的重组真核表达载体。方法:外源性热休克蛋白70基因连接入真核表达载体pDC315-EGFP中,转化大肠杆菌感受态细胞,重组真核表达载体外源基因测序及PCR检测鉴定阳性克隆后转染人胚肾293细胞,荧光显微镜及western blot鉴定外源基因在细胞中的表达。结果与结论:经PCR和测序证实外源性人热休克蛋白70基因正确重组入真核表达载体pDC315-EGFP,转染293细胞后荧光显微镜观察到绿色荧光蛋白GFP的表达,Western blot检测到热休克蛋白70在293细胞中有效表达。
BACKGROUND： Heat shock protein 70 （Hsp70） has attracted widespread attention by domestic and foreign scholars for the role of tumor immunity in recent years. However, the commonly used methods, such as heat stress and ischemic preconditioning, have disadvantages in inducing exogenous recombinant Hsp70 gene expression. OBJECTIVE： To construct a recombinant eukaryotic expression vector which expresses human HSP70 gene. METHODS： The exogenous Hsp70 gene was connected to eukaryotic expression vector pDC315-EGFP. Then it was transformed to E. coli competent cells. The positive clone was evaluated by gene sequencing and PCR detection and then was transfected to human embryo kidney 293 cells. The exogenous gene expression was tested by fluorescence microscope and Western blot. RESULTS AND CONCLUSION： It was confirmed by PCR and sequencing identification analysis that the target gene was cloned correctly to the eukaryotic expression vector. The expression of GFP could be observed by fluorescence microscope and western blot, which indicated that Hsp70 could be efficiently expressed in human embryo kidney 293 cells. Recombinant eukaryotic expression pDC315-EGFP-Hsp70 was constructed successfully.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第20期3674-3677,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家科技部"十五"863计划项目(2007AA021104)
课题名称:Hsp70
GM-CSF双修饰DU145
PC3激素非依赖性前列腺癌细胞株全细胞疫苗的研制
中南大学研究生学位论文创新基金
课题名称:Hsp70
GM-CSF双修饰DU145
PC3激素非依赖性前列腺癌细胞株全细胞疫苗的研制~~
