摘要
目的制备高纯度的人PPARγ1LBD融合蛋白,为筛选其配体奠定基础。方法以人脂肪组织总RNA为模板,利用RT-PCR方法扩增出人PPARγ1LBD基因的cDNA片段,将其插入表达载体pMAL-p2X的麦芽糖结合蛋白(maltose binding protein,MBP)编码基因malE的下游,转化宿主菌E.coli.TB1。并对重组质粒在大肠杆菌TB1中的表达条件进行了优化。结果经BamHⅠ和HindⅢ双酶切,获得分子量为909bpDNA条带;当诱导剂IPTG浓度为0.4mmol.L-1,30℃振荡诱导培养6h时,重组菌TB1高效表达MBP-PPARγ1LBD融合蛋白,表达量可达胞质总蛋白的38.54%。结论成功构建了能在TB1中高效表达人PPARγ1LBD融合蛋白的重组质粒,获得了具有高生物活性的融合蛋白。
Aim To obtain high pure PPARγ1 LBD fusion protein. Methods A cDNA encoding ligand binding domain （LBD） of PPARγ1 was amplified by RT-PCR from human fatty tissue and the product was inserted into the downstream of the malE gene in the vector pMAL-p2X, which encoded maltosebinding protein （MBP）. The recombinant plasmid containing MBP-PPARγ1 gene was transformed into E. coll. TB1 and the expression conditions of the recombinant strain were optimized. Resuits The DNA strap of MW（909 bp） was presented after recombinant plasmid was digested by Hind Ⅲ and BamH Ⅰ. The high efficient expression of MBP-PPARγ1 fusion protein in TB1 cells was observed with 38.54% product of the total cytoplasm proteins when 0. 4 mmol·L^-1 IPTG and 6 h incubation were taken at 30℃. Conclusion The recombinant vector was successfully constructed. It could high efficiently express PPARγ1 LBD fusion protein in TB1 cells and obtain the PPARγ1 LBD fusion protein with high bioactivity.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2008年第9期1254-1257,共4页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No30572353)
