人MUC1与GM-CSF基因融合真核表达质粒的构建与表达  

Construction and expression of eukaryotic coexpression plasmid containing human MUC1 gene and GM-CSF gene

作　　者：袁时芳[1] 师长宏[2] 晏伟[3] 姚青[1] 李南林[1] 王廷[1] 王岭[1] 张英起[4] 

机构地区：[1]第四军医大学西京医院血管内分泌外科,陕西西安710032 [2]第四军医大学实验动物中心,陕西西安710032 [3]第四军医大学病理学教研室,陕西西安710032 [4]第四军医大学生物技术中心,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2007年第1期18-20,24,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30571802);陕西省自然科学基金资助项目(2004C2-36)

摘　　要：目的:构建人MUC1重复序列串联体基因与GM-CSF基因重组的真核共表达质粒pcDNA3.1(+)-MUC1-GM-CSF,并观察重组质粒在COS-7细胞中的表达。方法:将信号肽及编码MUC1基因片段合成、合成的片段经退火等方法获得MUC1基因重复序列串联体,酶切鉴定及序列分析后,与GM-CSF基因克隆入pcDNA3.1(+)真核表达载体中,构建真核共表达质粒pcDNA3.1(+)-MUC1-GM-CSF。以重组质粒转染COS-7细胞,通过间接免疫荧光法及ELISA检测目的基因的表达。结果:酶切鉴定及序列分析表明,重组质粒含有人MUC1重复序列串联体与GM-CSF的融合基因,在COS-7细胞中可检测到MUC1表达,重组质粒免疫小鼠可诱导产生抗GM-CSF抗体。结论:人MUC1重复序列串联体与GM-CSF基因重组的真核共表达质粒的成功构建,为对其免疫原性、生物学特性及免疫治疗的进一步研究奠定了基础。AIM： To construct an eukaryotic coexpression plasmid containing the coding region of human MUC1 tandem repeats gene and GM-CSF gene and to identify its expression in COS-7 cell. METHODS： MUC1 tandem repeats gene was obtained by synthesizing the segments. After identified by restriction endonuclease digestion analysis and DNA sequencing, MUC1 tandem repeats gene and GM-CSF gene were cloned into eukaryotic expression vector pcDNA3.1（ ＋ ） to construct recombinant plasmid pcDNA 3.1 （ ＋ ）-MUC1-GM-CSF. Then the recombinant plasmid was transfected into COS-7 cell by electroporation and the expression of target gene was detected by immunoflourescence and ELISA. RESULTS： Restriction analysis and DNA sequencing showed that the recombinant plasmid contained the coding region of human MUC1 tandem repeats gene and GM- CSF gene. The expression of MUC1 and GM-CSF was detected. CONCLUSION： The suuessful construction and expression of recombinant plasmid pcDNA3.1 （ ＋ ）-MUC1- GM-CSF lay a foundation for further development of DNA vaccine against breast cancer.

关 键 词：基因工程 MUC1 GM-CSF 共表达载体 COS-7细胞 

分 类 号：Q786[生物学—分子生物学]