【摘要】 目的　通过对Aβ多肽基因进行重组克隆 ,构建质粒和高效表达的菌株 ,并研究表达产物快速高效的纯化回收方法。方法　采用IMPACT TWIN表达系统构建Intein Aβ重组基因表达质粒 ,将该质粒转化至BL2 1(DE3) ,筛选高效表达菌株 ;表达产物经ChitinBeads亲和层析柱分离纯化 ;SDS PAGE电泳 ,毛细管区带电泳及免疫印迹法等鉴定。结果　重组的Intein Aβ基因在BL2 1中获得高效表达 ,筛选出稳定高效表达的BL2 1(DE3)细胞株 ;融合蛋白表达量可达全菌蛋白的 6 0 % ;表达产物经ChitinBeads亲和层析纯化后 ,其纯度可达 98%以上 ,并具有与Aβ多肽相同的分子量及免疫学活性。结论　Aβ多肽基因在IMPACT TWIN系统中可获得高效表达 ,利用ChitinBeads亲和层析方法可将表达的Aβ多肽进行快速、简便、无酶化的高效分离纯化。更多还原

【Abstract】 AIM To construct overexpressed plasmid and strain by recombining and cloning Aβ peptide gene and study the approach of effectively and rapidly purifying expression product. METHODS The recombinant plasmid of Intein-Aβ was constructed by cloning Aβ gene into expression vector pTWIN1(IMPACT-TWIN System). The vector was then introduced into BL21(DE3)and the overexpressed strain was screened. The expression product was purified by affinity chromatograghy of Chitin Beads. Expressed Aβ peptide was analysed by SDS-PAGE gel, CZE and Western blot. RESULTS Recombinant pTWIN1 plasmid containing Intein-Aβ gene was overexpressed in E.coli and expressed fusion protein reached 60% of total bacteria protein. Stable and overexpressive BL21(DE3)strain was obtained. The expression product from affinity chromatograghy reached a purity of 98% and has the same MW and immune activity as Aβpeptide. CONCLUSIONS Overexpression of Aβ peptide gene and purification of Aβ peptide can be achieved by using the IMPACT-TWIN system. The expressed Aβ peptide can be easily, rapidly and effectively purified by affinity chromatograghy without protease.更多还原