摘要
目的:克隆、表达内皮素1(endothlin1,ET1)串联体,提高ET1的免疫原性及其免疫机体后产生抗体的效价.方法:通过pGEX-4T-1载体对ET1的基因进行了串联,并在ET1串联基因下游引入GM-CSF基因,酶切鉴定及序列分析构建重组质粒;再将含有ET1串体与GM-CSF的融合基因连接至真核表达载体pcDNA3.1(+)中,同样经过酶切鉴定及序列测定证实;并对构建表达载体转染COS-7细胞,应用间接免疫荧光法及ELISA检测表明目的基因在COS-7细胞中的表达情况.结果:正确构建了ET1重复序列4串体基因与GM-CSF基因重组的克隆载体pGEX-ET1④-GM-CSF以及真核共表达质粒pcDNA3.1-ET1④-GM-CSF;间接免疫荧光法及ELISA检测表明目的基因ET1和GM-CSF在COS-7细胞中都得到了表达.结论:内皮素1串联体的构建及其在COS-7细胞中的成功表达将对ET1的免疫学特性、核酸疫苗的研制以及相关自身疾病免疫治疗的进一步研究奠定基础.
AIM： To clone and express the tandem endothelinl （ ET1 ） gene of 4 repeats, to increase the immunogenicity of ET and to produce higher levels of ET1 antibody. METHODS： The tandem ET1 gene of 4 repeats, with the adjuvant gene GM-CSF were constituted by pGEX-4T-1 and cloned into eukaryotic expression vector pcDNA3.1 （＋）[ pcDNA-ETI④-GM-CSF]. The recombinant plasmid pcDNA3. 1-ET1④-GM-CSF was transfected into COS-7 cells by electroporation and the expression of target gene ET1 ④ and GM-CSF was assessed by immunofluorescence and ELISA. RESULTS ：The recombinant plasmid was successfully transfected into COS-7 cells by electroporation and the expression of target gene ET1 ④ and GM-CSF was demonstrated by immunofluorescence and ELISA. ELISA analyses showed that expressed fusion protein had immunogenicity of ET1 and produced the higher titer of GM-CSF in the transfected COS-7 ceils. CONCLUSION： The result of this research is helpful to study the relationships between ET1 and its relative proteins, and more helpful to lay the foundation for further development of DNA vaccine against autoimmunity diseases.
出处
《第四军医大学学报》
北大核心
2008年第14期1267-1270,共4页
Journal of the Fourth Military Medical University
