重组人血小板生成素在大肠杆菌中表达的研究

采用化学法全合成了编码人血小板生成素（ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ，ＴＰＯ）成熟肽Ｎ端１５３氨基酸的基因序列，构建基于该合成基因的表达质粒，结果以谷胱甘肽转硫酶－ＴＰＯ１５３（ＧＳＴ－ＴＰＯ１５３）融合蛋白的方式获得了占全菌蛋白４０％的高效表达．进一步采用ＰＣＲ方法分别对ＴＰＯ合成基因及ＴＰＯｃＤＮＡ的翻译起始区（ＴＩＲ）序列进行定点突变，以降低这一区域的Ｇ－Ｃ含量．将突变序列分别插入到ｐＢＶ２２０表达载体中，重组质粒在转化大肠杆菌ＪＭ１０９后，均获得了表达，其中ＴＩＲ区突变后的合成基因表达产物约占全菌蛋白的１５％．为研究基因下游结构对表达的影响，在不改变氨基酸组成的基础上，构建了ＴＰＯ合成基因与ＴＰＯｃＤＮＡ的杂合序列表达质粒．研究结果表明翻译起始效率是影响ｒｈ－ＴＰＯ在大肠杆菌中表达的重要因素之一，同时基因下游序列的组成对表达水平也会产生影响．

Study on the Expression of Recombinant Human Thrombopoietin in Escherichia coli

Several expression plasmids based on the TPO cDNA sequences were initially constructed,but no detectable expression was observed.Then the gene coding N terminal 1 153 amino acids of TPO mature peptide was chemically synthesized according to the E.coli preferred codons,and the resultant synthetic TPO153 gene was subcloned into several expression vectors,among which high level expression at 40% of total cellular proteins was achieved as GST TPO153 fusion protein.To obtain non fusion expression of TPO in E.coli ,a PCR based mutagenesis of the translation initiation region(TIR) was performed,and expression plasmids based on P RP L promoter were constructed,after transformation of E.coli JM109,the reconstructed synthetic TPO153 gene was expressed at the level of about 15% of the total cellular proteins.These results indicate that one of the most important factors that affect expression of rh TPO in E.coli is the efficiency of translation initiation.Moreover,three expression plasmids based on the hybrid sequences of the synthetic TPO gene and the TPO cDNAs were constructed,and the results indicated that the composition of downstream sequence could also influence the expression of rh TPO in E.coli to some extent.