You Leiming, Weng Haibo, Chen Zhankuan, Wang Aiping, Xu Weihua, Wang Minge, Dong Ziming
Department of Bioengineering, Zhengzhou University, 100 Kexue Road, Zhengzhou, People's Republic of China.
Mol Biol Rep. 2009 Sep;36(7):1793-8. doi: 10.1007/s11033-008-9382-5. Epub 2008 Nov 2.
A novel vector for direct PCR fragments cloning by positive selection, pBN, was constructed based on the lethal barnase from Bacillus amyloliquefaciens. Barnase was modified by inserting an additional insert at a pivotal Ile-54 site, which could take crucial affect on protein structure and absolute activity. The lacZ' expressing cassette of pUC19 was replaced by the modified barnase under the NptIII promoter. This novel vector could exist in large quantities as pUC19 in E. coli hosts. For the direct cloning PCR fragments, the positive selective vector was prepared by linearizing pBN with EcoRV to cut off the additional insert. PCR fragments with different length were prepared to verify this vector by ligation with this vector. The results showed that this positive selective vector for PCR fragment cloning was higher efficient and more convenient in manipulation than previous positive vectors.
基于解淀粉芽孢杆菌的致死性核酸酶Barnase构建了一种用于通过阳性选择直接克隆PCR片段的新型载体pBN。通过在关键的Ile-54位点插入一个额外的插入片段对Barnase进行修饰，这可能对蛋白质结构和绝对活性产生关键影响。在NptIII启动子的控制下，用修饰后的Barnase取代了pUC19的lacZ'表达盒。这种新型载体在大肠杆菌宿主中可以像pUC19一样大量存在。为了直接克隆PCR片段，通过用EcoRV线性化pBN以切除额外的插入片段来制备阳性选择载体。制备不同长度的PCR片段，通过与该载体连接来验证该载体。结果表明，这种用于PCR片段克隆的阳性选择载体比以前的阳性载体效率更高且操作更方便。
