用 PCR方法构建了一个不能形成二聚体的 C端缺失半胱氨酸 57的 h ITF突变体 .将其克隆到大肠杆菌表达载体 p GEX- 4T- 1中 ,ITPG诱导表达 ,融合蛋白经 Glutathione- Sepharose 4B亲和层析 ,凝血酶酶切和 Sephacryl S 1 0 0纯化 ,得到突变体蛋白 .SDS- PAGE,氨基酸组成 ,飞行质谱 ,N端氨基酸序列测定结果与期望值一致 .研究表明突变体的生物学活性有所降低 .对胃蛋白酶作用的稳定性降低 .
Abstract
A mutant that deleted Cys57 of human intestinal trefoil factor was construcuted through PCR amplification and expressed as fusion protein with GST in E.coli. The fusion protein was purified through three steps,Glutathione Sepharose 4B affinity chromatography,thrombin digestion and gel filtration on Sephacryl S 100.The results of SDS PAGE,mass spectrometry,amino acid composition and sequence of seven amino acids at the N terminus of the expressed protein were as expected.The biological activity of the mutant was much lower than that of hITF due to its sensitivity to pepsin digestion.
关键词
小肠三叶因子 /
突变体 /
生物活性
{{custom_keyword}} /
Key words
Intestinal trefoil factor /
Mutant /
Bioactivity
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
1 Babyatsk M W,Debeaumont M,Thim L,Podosky D K.Oraltrefoil peptides protect against ethanol-and indomethacin-induced gastric injury in rats.Gastroenterology. 1996,110 :489～ 497
2 口如琴 ,王蔚 ,李令媛 ,茹炳根 .小肠三叶因子对盐酸诱导的大鼠胃溃疡的预防与治疗作用 .中国药理学通报 ( Kou Ru-qin,WangWei,L i L ing-yuan,Ru Bing-gen.Precautionary and therapeuticeffect of recombinant human intestinal trefoil factor onhydrochloric acid-induced gastric ulcer in rats.Chin PharmacolBull) ,2 0 0 0 ,16( 2 ) :178～ 181
3 Thim L .A new family of growth factor-like peptides trefoildisulphide loop structures as a common feature in breastcancerassociated peptide ( p S2 ) ,pancreatic spasmolytic polypeptide( PSP) and frog skin peptides( spasmolysins) .F EBS lett,1989,2 5 :85～ 90
4 Suemori H,L ynch-devaney K,Podosky D K.Identification andcharacterization of rat intestinal trefoil factor:tissue-and cell-specific member of the trefoil protein family.Proc N atl AcadSci USA,1991,88:110 17～ 110 2 1
5 Chinery R,Bates P A,Amitabha D,F reem ont P S.Characterization of the single copy trefoil peptides intestinaltrefoil factor and p S2 and their ability to form covalentdimers.F EBS L ett,1995 ,3 5 7:5 0～ 5 4
6 王蔚 ,口如琴 ,李令媛 ,茹炳根 .人小肠三叶因子在大肠杆菌中的表达研究 .中国生物化学与分子生物学报 ( Wang Wei,Kou Ru-qin,Li Ling-yuan,Ru Bing-gen.Clone of human intestinaltrefoil factor in E.coli.Chin J Biochem Mol Biol) ,1999,15 ( 5 ) :72 4～ 72 8
7 口如琴 ,王蔚 ,李令媛 ,茹炳根 .小肠三叶因子的理化性质及光谱学行为 .中国生物化学与分子生物学报 ( Kou Ru-qin,WangWei,Li L ing-yuan,Ru Bing-gen. Chemico-physical propertiesand spectra of recombinant human intestinal trefoil factor.ChinJ Biochem Mol Biol) ,1999,15 ( 5 ) :787～ 791
8 Playford R J,Marchbank T,Chinery R,Ruth E. Humanspasmolytic polypeptide is a cytoprotective agent thatstimulates cell migration. Gastroenterology,1995 ,10 8:10 8～116
9 张龙翔 ,张庭芳 ,李令媛主编 .生化实验方法和技术 .第 2版 .北京 :高等教育出版社 ( Zhang L ong-xiang,Zhang Ting-fang,L iL ing-yuan ed. Exp erimental Methods and Techniques inBiochemistry,2 nd ed.Beijing:Higher Education Press) ,1997:10 0～ 115
10 Taupin D R,Ori K,Shulkes A,Giraud A S. Conservedexpression of the intestinal trefoil factor in the human colonicadenoma-carcinoma sequence.Lab Invest,1994,74( 1) :2 5～ 3 2
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
PDF(292 KB)
