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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。
Objective To clone human thrombopoietin (hTPO) gene and express it in prokaryotic cells. Methods Total RNA was extracted from human embryonic liver and subjected to RT-PCR to obtain hTPO1 95 cDNA encoding amino terminal amino acid residues. The fragment was subcloned into the pGEM-Teasy cloning plasmid and the cloned plasmid was directly sequenced by the dideoxy chain termination method. Then, the hTPO1 95 cDNA was inserted into prokaryotic expression vector pET2 8-a and transformed into E. coli BLR2 1 (DE3) for expression. SDS-PAGE and Western blotting were used to detect the expression of hTPO1 95 cDNA. The resulting hTPO1 95 cDNA was exactly the same as reported in the literature. After induction, the hTPO gene was expressed in E. coli, and the target protein accounted for about 10% of the total bacterial proteins. The correct expression product was confirmed by immunoblotting. Conclusion The hTPO1 95 cDNA was obtained and expressed in prokaryotic cells. The truncated form of rhT PO1 95 was obtained.