人三叶因子3双结构域突变体的构建

利用基因工程方法在大肠杆菌-酶母穿梭质粒pPIC9K上构建了人三叶因子3(HumanTrefoilFactor3,hTFF3)双结构域突奕体基因，采用毕氏酵母表达系统对目的基因进行了分泌表达。经S-Sepharose,Q-Sepharose,SephacrylS-100纯化后得到突变体蛋白，主要以单体形式存在。SDS-PAGE表明重组蛋白分子量约为12000，并在Westernblotting印迹实验中证明能被抗hTFF3抗体所识别，N-端氨基酸测序与天然hTFF3-一致，质谱检测纯化的蛋白出现单体和双体两种形式。用重组蛋白对乙醇诱导的大鼠胃溃疡进行实验治疗表明hTFF3双体突变体与天然形成的双体蛋白具有相同的活力，并且活力高于单体形式的hTFF3。

Construction of double-trefoil domain mutant ofhumantrefoil eactor 3

Double-trefoil domain mutant of human trefoil factor 3 (hTFF3) was constructed through gene engineering and inserted into to pPIC9K plasmid, then expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein was purified through S-Sepharose, Q-Sepharose and Sephacryl S-100. Molecular weight of the expressed protein was about 12 000 identified by SDS-PAGE and Western-blotting. N-terminal amino acid analyses proved that the expressed hTFFE3 dimeric form mutant was identical to the native N-terminus. Two protein peaks corresponding to the monomeric and dimeric form of Di-hTFF3 mutant were identified by the mass spectrometry. The boiactivity of the mutant was the same as the native dimeric form of hTFF3, and higher than the of the monomeric mutant.