摘要
目的:构建含组氨酸标签的FMS样酪氨酸激酶3配体(Fms-like tyrosine kinase 3 ligand,Flt3L)胞外域蛋白的原核表达载体,并在大肠杆菌中进行表达。方法:依据Flt3L基因序列设计引物,以RT-PCR的方法从小鼠脾脏总RNA中克隆Flt3L基因并构建其胞外域原核表达载体,测序鉴定后在大肠杆菌BL21(DE3)菌株中诱导表达,用免疫印迹鉴定。结果:克隆到Flt3L编码区全长序列,经DNA测序证明与已报道的序列相同;构建羧基端带有His6标签的Flt3L胞外域蛋白的原核表达载体,转化大肠杆菌后SDS-PAGE分析显示在BL21(ED3)中获得较高表达;免疫印迹分析表明IPTG诱导后表达的Flt3L胞外域蛋白的相对分子质量(Mr)为19 000,与理论值相符,此蛋白与抗His6标签的抗体发生特异性反应。结论:成功克隆Flt3L基因,构建其胞外域蛋白的原核表达载体,并在大肠杆菌中获得表达。
Aim： To clone the eDNA of FMS-like tyrosine kinase 3 ligand （Flt3L） and to construct the prokaryotic expression vector for the Flt3 ligand extracellular domain fused with a His6 tag and express in Escherichia coli （E. coli）. Methods： The primers were designed according to the Flt3L gene, and the eDNA for Flt3L was cloned by RT-PCR from the mouse spleen total RNA and confirmed by DNA sequencing. The DNA fragment encoding Flt3L extracellular domain was amplified by PCR with the sequenceverified Flt3L eDNA as a template and then cloned into pET-3c vector. After confirmed by DNA sequencing once again, the prokaryotic expression vector was transformed into BL21 （DE3） strain. The recombinant protein was expressed in E. coli after IPTG induction. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. Results： DNA sequence analysis showed that the eDNA encoding Flt3L was cloned. The expression vector for the recombinant Flt3L extracellular domain was then constructed and verified by DNA sequencing. SDS-PAGE analysis revealed that the fusion protein was highly expressed in E. coli. Furthermore, Western blotting showed that the recombinant protein existed largely in the inclusion bodies. The fusion protein had a molecular weight of about 19 000, which is in accordance with the theoretical value. Conclusion： The cDNA of Flt3L was cloned and the prokaryotic expression vector for the fusion protein of Flt3L was constructed. The recombinant protein was highly expressed as the form of inclusion bodies in E. coli, which would facilitate the further study of soluble Flt3L.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2008年第4期331-335,共5页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家自然科学基金项目(30572199
30230350和30371651)
生物化学与分子生物学广东省重点学科资助项目
关键词
FMS样酪氨酸激酶3配体
胞外域
原核表达
组氨酸标签
FMS-like tyroslne kinase 3 ligand
extracellular domain
prokaryotic expression
hexahistidine tag
