摘要
低磷血症是人类磷代谢失常的常见疾病,成纤维细胞生长因子23 (fibroblast growth factor-23, FGF23)可以作用于肾曲小管减少肾脏对无机磷重吸收。保留C端73个氨基酸的FGF23C-tail作为FGF23的抑制剂,可以增加肾脏对无机磷重吸收,因此FGF23~(C-tail)在低磷血症的药物开发中显得尤为重要。本文首次将FGF23~(C-tail)与人血清白蛋白(human serum albumin, HSA)融合,并尝试在毕赤酵母表达系统制备表达。融合PCR构建重组基因,经克隆株的抗性筛选和表达量筛选,获得一株毕赤酵母高产量的表达菌株,摇瓶的表达量为43.7 mg·L~(-1)。5 L发酵罐的研究表明,表达量可高达265.6 mg·L~(-1)。经三步纯化步骤后,获得纯度大于95%的FGF23C-tail-HSA融合蛋白。经宜春学院伦理委员会批准,蛋白的体内大鼠实验显示, FGF23C-tail-HSA具有FGF23抑制剂的功能,且能显著地提高大鼠的血磷水平。本研究为FGF23C-tail-HSA生理学活性的深入研究和FGF23C-tail的药物开发提供基础。
Hypophosphatemia is a common metabolism disease in humans. Fibroblast growth factor 23(FGF23) inhibits phosphate reabsorption by targeting on the renal tubules. FGF23~(C-tail)contains 73 amino acids from C-terminus of FGF23, serves as an inhibitor of FGF23, and can increase phosphate reabsorption. Therefore,FGF23C-tailis an important drug for hypophosphatemia. In this paper, we constructed a fusion protein of FGF23~(C-tail) with HSA, and investigated the expression of the fusion protein in the Pichia pastoris system. The recombinant gene was constructed by fusion PCR. A high-yield strain was selected by G418 resistance and fermentation yield,and the expression yield was 43.7 mg · L~(-1)in flask. In 5 L fermenters, the highest expression yield could reach265.6 mg · L~(-1). FGF23C-tail-HSA could be used as an inhibitor for FGF23, and could significantly increase blood phosphorus levels in rats. The procedures for care and use of animals were approved by the Ethics Committee of YiChun University. This paper provided a basis research for further studying physiological activity of FGF23C-tailHSA.
引文
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