摘要
目的:通过获得编码血小板生成素(Tpo)成熟肽N端1~196个氨基酸的突变体cDNA在大肠杆菌JM109中的表达,为Tpo进一步的结构和功能研究提供材料来源。方法:用多聚酶链反应(PCR)及DNA重组技术,将PCR产物克隆到pUC19载体并测序,然后克隆到表达载体pMAL-c2上。结果:转化重组质粒pMAL-MBP/TpoM的大肠杆菌经异丙基-β-D-硫代半乳糖苷诱导4~5小时,SDS-PAGE分析显示融合蛋白(MBP/TpoM)的分子量约为6.3万。薄层扫描分析表明该蛋白占菌体总蛋白量的37%。结论:经PCR方法扩增的Tpo基因突变体可在大肠杆菌中高效表达,为Tpo突变体的进一步结构和功能研究以及Tpo抗体的制备打下了基础。
Objective:To obtain human thrombopoietin (Tpo)mutant cDNA encoding mature peptide N terminal 1~196 amino acids and investigate its expression in E.coli JM109.Methods:Polymerase chain reaction and DNA recombination techniques were employed.PCR product was inserted into pUC19 vector and sequenced and then cloned into expression vector pMALc2.Results:E.coli JM109 cells with plasmid pMAL MBP/TpoM were induced by IPTG for 4~5 hours.SDSPAGE analysis showed that MBP/TpoM molecular weight is about 63000.Scanning analysis indicated that expressed protein accounts up to 37% of total E.coli proteins.Conclusion:The Tpo mutant of interest is successfully expressed in E.coli JM109 cells.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
1997年第12期634-637,共4页
Chinese Journal of Hematology