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利用PCR方法扩增炭疽杆菌噬菌体裂解酶 (γlysin)基因 ,克隆至大肠杆菌表达载体pET2 2b中 ,经菌落PCR筛选、序列测定和酶切鉴定证实表达载体pET2 2b -γlysin构建成功 ,并在EscherichiacoliBL2 1(DE3)中获得了高表达。目的蛋白约占菌体总蛋白的 4 0 % ,5L发酵罐中的产酶水平高达 15g L。菌体经超声破碎 ,制备无细胞抽提液 ,StreamlineSP和SPHP柱层析以及SephacrylS- 10 0凝胶过滤三步纯化 ,得到分子量为 2 7kD单一条带的目的蛋白 ,薄层扫描分析显示其纯度大于 95 %。目的蛋白的收率为 19 1% ,纯化倍数为 35 0。生物活性鉴定重组的γ噬菌体裂解酶具有特异性 :可快速裂解炭疽杆菌 ,比活为 14 0 0u mg左右 ;而对大肠杆菌、枯草杆菌及蜡样芽孢杆菌没有裂解活性。
The γlysin gene was amplified by PCR and cloned into E.coli expression vector pET2 2b. After colony PCR screening, sequence analysis and restriction enzyme digestion proved that the expression vector pET2 2b -γlysin was successfully constructed and expressed in Escherichia coli BL21 (DE3). The target protein accounts for about 40% of the total bacterial protein, 5L fermentor in the production of up to 15g L level. The bacterial cells were sonicated, and cell-free extracts were prepared. StreamlineSP and SPHP column chromatography and Sephacryl S-10 gel filtration three-step purification, the molecular weight of 27kD a single band of the target protein, thin layer scanning analysis showed that the purity Greater than 95%. The yield of the target protein was 19 1% and the purification fold was 35 0. Identification of biological activity of recombinant γ phage lyase specificity: rapid Bacillus anthracis, specific activity of about 14 0 0u mg; Escherichia coli, Bacillus subtilis and Bacillus cereus no lytic activity.