【摘要】 目的 :实现含人类胰岛素生长因子I(hIGF - 1)融合蛋白的高水平可溶性表达和纯化 ,应用factorXa获得非融合的hIGF - 1,并对提高目的融合蛋白对factorXa的敏感性进行探索。方法 :根据大肠杆菌偏爱密码子设计并合成编码 70个氨基酸的hIGF - 1基因 ,克隆到表达载体pET - 35b(+) ,实现hIGF - 1与纤维素结合域 (cellulosebindingdomain ,CBD)的融合表达 ,并在两者之间引入factorXa的识别位点 ,同时尝试在factorXa识别位点前引入 7个氨基酸的柔性短肽 (Gly -Thr-Gly -Gly-Gly -Ser -Gly) ,比较factorXa酶切效率。纤维素亲和层析纯化融合蛋白CBD -IGF ,经Westernblotting检测后 ,利用MTT法测定其促进NIH3T3细胞增殖的活性。结果 :可溶性融合蛋白CBD-IGF在大肠杆菌中高效表达 ;活性测定结果显示所纯化的融合蛋白具有促进 3T3细胞增殖的活性。在factorXa识别位点前引入柔性短肽后提高了融合蛋白对factorXa的敏感性。结论 :实现有活性的融合蛋白CBD -IGF在大肠杆菌中可溶性的高效表达 ,为高效制备hIGF - 1基因工程产品奠定了基础。此外 ,在factorXa识别位点前引入柔性短肽有效提高融合蛋白CBD -IGF对factorXa的敏感性。更多还原

【Abstract】 AIM: To express the synthesized human insul in like growth factor I (hIGF-1) gene in E.coli with high expression level a nd explore the way to increase the efficiency of factor Xa cleavage. METHODS: The gene of hIGF-1 was designed and synthesized accordi n g to the preference of E.coli. A fusion protein with a recognized site of fa ctor Xa between CBD (cellulose binding domain) and hIGF-1 was expressed and puri fied by cellulose affinity chromatography. MTT method was used to assay the bioa ctivity of CBD-IGF fusion protein. hIGF-1 was released by factor Xa. In order to improve the sensitivity of fusion protein to factor Xa, the short flexible pept ide (Gly-Thr-Gly- Gly-Gly-Ser-Gly) was added before the recognized site of fac tor Xa. RESULTS: SDS-PAGE results indicated that the CBD-IGF fusion prot ein was expressed and purified . Biological assay results indicated CBD-IGF fusi on protein could promote the growth of NIH3T3 cell. The short flexible peptide (Gly-Thr-Gly-Gly-Gly-Ser-Gly), which was added before the recognized site of f actor Xa, improved the sensitivity of fusion protein to factor Xa. CONCLUSION: CBD-IGF fusion protein with bioactivite are expresse d and purified. The amio acid sequences changes between the site recognize of fa ctor Xa can help to improve the cleavage efficiency of Factor Xa.更多还原