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目的为了提高促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)的稳定性,针对ACTH中多种血液蛋白酶的作用位点,制备特定氨基酸位点突变(E5D、F7L、G10A及Y2S)基因重组长效人ACTH,分析比较哪一种点突变基因重组ACTH保留了天然野生型的生理活性。方法采用ELISA和q PCR等方法比较了4种突变型ATCH与野生型ACTH对体外培养的小鼠肾上腺皮质Y1细胞分泌糖皮质激素(glucocorticoids,GC)水平的影响,以及它们对ACTH合成的主要限速酶基因(STAR、P450-scc、3β-HSD)以及对STAR和P450-scc的合成发挥关键调节作用的类固醇合成因子1(SF-1)的转录水平的影响。结果 E5D及Y2S突变型ACTH对Y1细胞分泌GC以及对SF-1、STAR、P450-scc、3β-HSD等基因表达的影响与野生型ACTH比较,没有显著性差异。结论一级结构修饰的E5D和Y2S突变型ACTH的生理活性与野生型ACTH相当。
Objective To improve the stability of adrenocorticotropic hormone (ACTH), a series of recombinant human long-acting human ACTH (E5D, F7L, G10A and Y2S) gene were prepared according to the site of action of many blood proteases in ACTH , Analysis and comparison of which point mutation gene recombinant ACTH retains the natural wild-type physiological activity. Methods The effects of four mutant ATCH and wild-type ACTH on the secretion of glucocorticoids (GCs) from mouse adrenal cortex Y1 cells in vitro were compared by ELISA and q PCR. The main limitation of ACTH synthesis Speed enzyme genes (STAR, P450-scc, 3β-HSD) and steroid synthesis factor 1 (SF-1), which plays a key regulatory role in the synthesis of STAR and P450-scc. Results The effect of E5D and Y2S mutant ACTH on the secretion of GC and the gene expression of SF-1, STAR, P450-scc and 3β-HSD in Y1 cells was not significantly different from that of wild-type ACTH. Conclusion The primary structure-modified E5D and Y2S mutant ACTH have the same physiological activities as wild-type ACTH.