【摘要】 大蹼铃蟾三叶因子Bm-TFF2具有较人TFF2更强的促细胞迁移和抗凋亡活性。该研究利用RT-PCR方法扩增得到野生型Bm-TFF2的基因,然后分别构建N端、C端和分子中两个精氨酸突变的突变体,最后连接表达载体产生pET32a(+)/Bm-TFF2突变型重组质粒,转入大肠杆菌中,经37℃培养,IPTG诱导,其融合蛋白主要存在于包涵体中,用组氨酸标签的亲合柱纯化溶解后的包涵体上清,进一步用RP-HPLC纯化得到硫氧还蛋白(TRX)/Bm-TFF2突变型融合蛋白。通过SDS-PAGE和Westernblotting检测分析其纯度和特异性。最终,从1L培养基中得到20mg纯度为95%的三种重组突变型融合蛋白。三种突变型重组蛋白都具有剂量依赖性的促细胞迁移活性,并且其活性无显著差异。该研究为进一步研究Bm-TFF2结构和功能的关系以及揭示其作用的分子机制奠定了基础。更多还原

【Abstract】 Bm-TFF2,a trefoil factor from the large-webbed bell toad(Bombina maxima),can stimulate cell migration and inhibit cell apoptosis.To study the structure-function relationship of Bm-TFF2,we constructed wild-type and mutated Bm-TFF2 plasmids and expressed recombinant proteins in E.coli.The wild-type Bm-TFF2 gene encoding mature peptide was obtained by RT-PCR,while the N-terminal,C-terminal and two arginine mutated Bm-TFF2 clones were constructed,and ligated into pET-32a(+) expression vectors.The fusion proteins were induced by IPTG at 37℃.The mutant Bm-TFF2 fusion proteins expressed mainly in the inclusion bodies.The mutant(TRX)/Bm-TFF2 could be purified by using Ni2+-chelating chromatography and reverse-phase HPLC from the inclusion body supernatant.The fusion proteins were analyzed by SDS-PAGE and Western blotting.The yield of mutant Bm-TFF2 fusion proteins of above 95% purity was about 20 mg/L.All three recombinant mutant proteins can promote the migration of AGS cells in a dose-dependent manner with no obvious activity difference.更多还原