血小板生成素基因的克隆及在原核细胞的表达

目的　克隆人血小板生成素 (hTPO)基因 ,并使其在原核细胞中获得表达。方法　先从人胚胎肝脏中提取总RNA ,进行RT -PCR获得编码氨基端 1 95个氨基酸残基的hTPO1 95cDNA。将该片段亚克隆至pGEM -Teasy克隆质粒中 ,以双脱氧链末端终止法对克隆质粒直接测序。接着将hTPO1 95cDNA插入到原核表达质粒 pET2 8-a中 ,转化大肠杆菌BLR2 1 (DE3) ,进行诱导表达 ,以SDS -PAGE方法及免疫印迹法进行检测。结果　得到的hTPO1 95cDNA与文献报道的序列完全一致。经诱导后hTPO基因在大肠杆菌中获得表达 ,目的蛋白占总菌体蛋白约 1 0 % ,以免疫印迹法证实表达产物正确。结论　得到hTPO1 95cDNA ,并在原核细胞中获得表达 ,得到截短形式的rhT PO1 95。

Cloning of human thrombopoietin and it's expression in E. coli

Objective Expression of human thrombopoietin(hTPO) in prokaryotic system. Methods The hTPO 195 ?cDNA coding N terminal 195 amino acid residues was cloned from the fetal liver mRNA by means of RT PCR and subcloned into the cloning plasmid pGEM T/easy.The recombinant plasmids were sequenced.The hTPO 195 cDNA was inserted into the expression plasmid pET28 a and expressed in E.coli. The recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS PAGE) and Western blotting.Results The sequence of the fragment is identical to that reported. The recombinant protein was about 10 percent of the total bacterial protein and confirmed by Western blotting. Conclusion The hTPO 195 ?cDNA was obtained and expressed in E.coli.