基因工程制备hitf及其对肠粘膜保护作用的研究-儿科学消化专业论文.docxVIP

间给予实验动物重组hlTF，观察不同浓度hITF在整体水平对肠粘膜 间给予实验动物重组hlTF，观察不同浓度hITF在整体水平对肠粘膜 的预防性保护作用以及促进肠粘膜损伤后修复的作用。 在5L发酵罐中获得发酵液约3．5L，上清液约2．5L，总纯化得率 为3j一45％，共获得目的蛋白250mg，纯度96％，蛋白表达水平为 73．33mg／L。体外实验结果表明，在10一～10。浓度范围内重组hITF 能够促进肠上皮细胞的迁移，但是对肠上皮细胞的光镜一卜形态和肠，h 皮细胞的增殖功能影响不显著。整体水平实验结果显示，在肠粘膜损 伤之前或之后给予不同浓度的外源性hlTF的实验组，大鼠肠粘膜损 伤程度明显较对照组轻，大体损伤评分和病理组织学评分均明显低于 对照组，而且实验组的髓过氧化物酶活性也明显低于对照组。表明 hITF既有对肠粘膜受损的预防性保护作用，又有促进肠粘膜损伤后 修复的作用。 本实验为大规模：[业化生产重组h!TF奠定了基础，同时对hITF 促进肠粘膜修复的作用和细胞生物学机制进行了探讨。 关键词人小肠三叶因子，重组hITF，毕赤酵母，发酵表达，肠 粘膜，损伤修复 II ABSTRACTHIGH ABSTRACT HIGH LEVEL EXPRES S10N 0F RECOMBINANT HUMAN INTESTnqAL TREFOIL FACTOR IN PICHIA PASTORIS AND ITS PROTECTIoN AGAINST日、『TESTINAL MUCOSAL DAMAGE ABSTRACT Diarrhea disease was a common and even frequently—occurring disease in pediatrics．However,most of the present therapeutic measures could only partially alleviate symptoms and leave the injured intestinal mucous unrepaired，which led to therapy delay or even worsen to chronic disease，together with severe dystrophy,growth retardation or loss of labor abilities．The early repair of intestinal mucosal injuries contributed to restriction of fluid flow,prevention from loss of electrolytes and suppression allergy reaction caused by exterior antigen pervading into the local or whole body．As far as the early repair of intestinal mucous injuries was concerned，the molecular mechanism still had to be explored． Composed of 59 amino acid residues，Intestinal trefoil factor was expressed and secreted by intestinal mucous．A unique three—looped trefoil motif conferred ITF the integrity of structures and functions in the digestive canal．Because ITF had such a low densities in the tissue that it was very difficult to extract it from the tissue，we try to get ITF by gene engineering，So that with plenty of ITE we explored its protection to III 垒!!!!!竺! 垒!!!!!竺! 一—— intestinal nlucous．The expression plasmid pPIC9／hITF containing AOXl promotor and the sequences of secreting signal peptides was transformed into the yeast cells．Then thro