摘要
目的克隆人白细胞介素(IL)15全长cDNA,并在大肠杆菌中表达。方法从人外周血分离的淋巴细胞中提取总RNA,通过RT-PCR扩增出hIL-15全长cDNA。构建到原核表达载体pET28a(+)中,通过卡那霉素平板筛选及PCR鉴定,挑出阳性克隆进行扩增,DNA测序正确后,转化到大肠杆菌BL21(DE3)中,扩增后IPTG诱导表达。通过SDS-PAEG及Western-blot方法鉴定表达的融合蛋白。结果成功构建人IL-15表达载体,并表达于大肠杆菌中。SDS-PAEG及Western-blot分析显示表达的融合蛋白分子量为16·1ku,与理论值相符。结论获得了hIL-15融合蛋白,为hIL-15单克隆抗体的制备及其功能研究奠定了基础。
Objective To clone full length cDNA of human Interleukin-15 （hIL-15） and express it in E coli. Methods Total RNA was isolated from peripheral lymphocyte, hlL-15 cDNA was amplified using RT-PCR. The expression plasmid pET28a（ ＋ ）-hiLl5 was constructed by inserting IL-15 cDNA into pET28a（ ＋ ）and then was transformed into BL21 （DE3）. Expression of hlL-15 was induced by IPTG at 37℃ for 6h. The fusion protein was analyzed using SDS-PAGE and Western-blot. Results hIL-15 was cloned and expressed in E coli successfully. SDS-PAGE analysis showed the molecular weight of the IL-15 was about 16. 1 ku. Conclusion The rhlL-15 was obtained by prokaryotic expression, which lays the foundation for study of its function.
出处
《安徽医科大学学报》
CAS
北大核心
2006年第5期491-493,共3页
Acta Universitatis Medicinalis Anhui
基金
安徽省自然科学基金资助(编号:050430604)
