炭疽杆菌噬菌体裂解酶基因在大肠杆菌中的表达及鉴定

Expression and Bioactive Characterization of Bacteriophage Lysin Gene of Bacil lus anthracis in Escherichia coli

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 lysin gene of Bacillus anthracis-diagnosing bacteriophage,obtaine d by PCR amplification,was cloned into the Escherichia coli exepression vect or p ET22b which has been cleaved by EcoRⅠ and NdeⅠ.The recombinant vector pET22b-γ l ysin was verified to be correctly constructed by PCR, sequencing and enzyme dige stion,and highly expressed in E.coli BL21(DE3), which accounted for about 40 petcent of total protein in E.coli BL21(DE3),while in the 5L fermentor the ex pres sion level reached 15g/L.After expression, disruption and purification with thre e-step chromatography, Streamline SP, SP HP and Sephacryl S-100,the recombinan t γ lysin was finally obtained with purity of higher than 95 percent as determ ine d by gel scan. The final yield following SP HP was 19.1 percent,with a greater - than-350-fold increase in specific activity.The pure enzyme has been shown act iv e to Bacillus anthracis,and not to E.coli,Bacillus subtilis and Bacill us cereus. Its specific activity was about 1400 u/mg.