优化mRNA翻译起始区提高人粒－巨噬细胞集落刺激因子在E．coli中的表达

人粒－巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）是一种重要的造血生长因子．利用基因重组技术构建两个ｈＧＭ－ＣＳＦ的Ｅ．ｃｏｌｉ表达菌株，一个为在不改变氨基酸顺序的前提下，对ｍＲＮＡ翻译起始区核苷酸顺序进行优化突变（ｈＧＭ－ＣＳＦ（Ｍ）），另一个为未突变的对照（ｈＧＭ－ＣＳＦ（Ｎ））．经酶切电泳、ＤＮＡ测序、ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ等分析鉴定，证明两者均能表达特异性的１４．６ｋＤｈＧＭ－ＣＳＦ，但ｈＧＭ－ＣＳＦ（Ｍ）的表达水平较ｈＧＭ－ＣＳＦ（Ｎ）提高了１．２６倍，占菌体总蛋白的１６．９％．ｍＲＮＡ翻译起始区二级结构预测分析表明，优化突变后生成自由能ΔＧ从原来的－１０．２提高至－９．４Ｋｃａｌ，ＡＵＧ从部分配对状态变为非配对状态．

Improving th Expression of Human Granulocyte-Macrophage Colony Stimulating Factor in E.coli by Optimizing the mRNA Translation Initiation Region

Human granulocyte-macrophage colony stimulating factor(hGM-CSF) is an important hematopoietic growth factor.Two E.coli strains expressing hGM-CSF have been constructed by DNA recombination techniques.One (hGM-CSF (M)) was that the nucleotides distributing over translation initiation region (TIR) was mutated while the intact amino acid coding sequence was kept unchanged,the other(hGM-CSF (N) was not-mutated and was served as control. Characterized by restriction analysis,DNA sequencing,SDS-PAGE and Western blotting,it was proved that both strains were able to express a 14. 6kD mature hGM-CSF, protein, but the expressive level of hGM-CSF (M) was raised 1. 26 times in comparison with hGM-CSF (N) ,with the yeild up to 16.9% of the total proteins of the bacterial lysis. Result of the predictive analysis of mRNAsecondary structure showed that the ΔG was elevated from -10.2 to -9. 4 Kcal and AUGcodon was released from the involvement of partial bases paring after performing the mutation.