Shen Yi-Jun, Pan Wei, Xu Rong, Pan Xin, Wang Luo-Chun, Wang Zheng, Cao Feng, Tan Jing-Wei, Wu Jin-Song, Wu Fang, Liu Yan-Jun
Department of Microbiology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Jan;20(1):43-8.
To construct the combined site-directed random mutation library of recombinant human Lymphotoxin (rhLT) for in vitro molecular evolution study, and to study the structure and function relationship. The random point mutations at the sites of 46,106 and 130 were individually generated by overlap PCR amplification with the random nucleotide primers. The three point mutations were combined and cloned into pMD-18T vector to construct the combined mutation library. DNA sequencing was used to evaluate the diversity and randomness of the mutation sites. The combined mutation library was re-engineered, inserted in prokaryotic expression vector pBV220, transformed and expressed in Escherichia coli strain DH5alpha. The biological activity of some of the mutants was tested in 1929 mouse fibroblast cells. As much as 1.5 x 10(5) clones were obtained, which represents 4.5 times of the complete mutation libraries at 99% confidence. Sequencing 50 clones revealed no obvious bias in the nucleotide and amino acid mutations at the sites. Among the 30 expressed samples underwent for the bioassay, 70% (21 samples) were inactive, 23.3% (7 samples) had lower activity than rhLT, the remaining 6.7% (2 samples) had higher activity than rhLT. The combined site-directed random mutation library of rhLT has been constructed successfully. In combination with phase display, the library is ready for in vitro molecular evolution study.
构建重组人淋巴毒素（rhLT）的组合定点随机突变文库用于体外分子进化研究，并探讨其结构与功能的关系。利用随机核苷酸引物通过重叠PCR扩增分别在46、106和130位点产生随机点突变。将这三个点突变组合并克隆到pMD-18T载体中构建组合突变文库。采用DNA测序评估突变位点的多样性和随机性。对组合突变文库进行改造，插入原核表达载体pBV220，转化并在大肠杆菌DH5α菌株中表达。在1929小鼠成纤维细胞中检测部分突变体的生物学活性。获得了多达1.5×10⁵个克隆，在99%置信度下代表完整突变文库的4.5倍。对50个克隆进行测序，结果显示位点处的核苷酸和氨基酸突变无明显偏向性。在进行生物活性检测的30个表达样品中，70%（21个样品）无活性，23.3%（7个样品）活性低于rhLT，其余6.7%（2个样品）活性高于rhLT。成功构建了rhLT的组合定点随机突变文库。结合噬菌体展示，该文库可用于体外分子进化研究。
