摘要
目的:克隆人Runx3基因全长编码区的cDNA,进行鉴定和测序分析,并利用载体pQE30在大肠埃希菌(M15)原核表达人Runx3基因全长序列,为从转录水平探讨Runx3基因与免疫相关性疾病以及肿瘤发生发展的关系奠定基础。方法:MACS分离外周CD8+T细胞;采用RT-PCR方法从人外周血CD8+T细胞获得Runx3基因的cDNA,并连接pMD19-T载体导入大肠埃希菌DH5α,选择阳性克隆,应用M13F/R通用引物进行双向反应测序,以作序列鉴定。利用PCR技术从克隆载体pMD19-T/Runx3获得人Runx3的全长编码序列,亚克隆至原核表达载体pQE30,形成重组表达质粒pQE30/Runx3;酶切鉴定挑选阳性重组质粒转化大肠埃希菌M15,测序鉴定后经IPTG30℃诱导4h,SDS-PAGE电泳判断以包涵体形式存在的带有His标签的融合蛋白并进行Western blot鉴定。结果:扩增获得的Runx3基因CDS区全长1248bp,编码415个氨基酸残基,与GenBank中发表的序列完全一致。成功构建了原核表达载体pQE30/Runx3,并在工程菌M15中获得大量表达.结论:获得了人Runx3基因的克隆,并成功原核表达和制备出人Runx3融合蛋白。
AIM： Runx3, a type of Runt family member, plays an important role in immune regulation. In this study, we cloned and analyzed the cDNA encoding human Runx3 from T lymphocyte, expressed Runx3 protein in E. coli system, and studied the relation between Runx3 and some immune disorders or tumors. METHODS： The CD8＋ T were isolated from human peripheral blood with MACS, Runx3 cDNA was amplified by RT-PCR and cloned into pMD19-T vector, and recombinant was transformed into competent cells DH5α and recombinant sequencing were performed. The identical was subcloned into pQE30 vector and expressed in E. coli M15. The fusion protein was identi- fied by Western blot. RESULTS： The 1 248 bp fragment amplified by RT-PCR was the same as the anticipated one in size and encodes 415 amino acids. Runx3 protein was gained. CONCLUSION： Human Runx3 gene was cloned and expressed in E. coil system successfully, which brought a foundation for further research on its biological function.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2009年第2期107-110,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30710103093
30300169
30671984)
江苏大学大学生科研立项(06A081)
