重组人淋巴毒素随机点突变组合文库的构建  

Construction of Combined Site-directed Random Mutation Libraries of Recombinant Human Lymphotoxin

作　　者：沈毅珺[1] 潘卫[1] 徐容[2] 潘欣[1] 王罗春 王征 曹峰 谭靖伟 吴劲松 吴芳 刘彦君 

机构地区：[1]第二军医大学基础医学部微生物学教研室,上海200433 [2]安徽医科大学病理生理学教研室,合肥230032 [3]上海复旦张江生物医药股份有限公司,上海201203

出　　处：《生物工程学报》2004年第1期43-48,共6页Chinese Journal of Biotechnology

基　　金：国家"8 63"计划基金项目资助 (No .2 0 0 1AA2 15 0 5 1;2 0 0 2AA2Z3 3 0 9)~~

摘　　要：构建重组人淋巴毒素 (rhLT)随机点突变组合文库以进行体外分子进化及结构和功能的研究。应用含随机核苷酸序列的引物 ,通过OverlapPCR的方法分别对rhLT的 4 6、10 6和 130位氨基酸进行定点随机突变 ,获得各单点随机突变体库。通过基因操作将这三个单点随机突变体库拼接并克隆于pMD_18T载体建立三点组合突变体文库 ,DNA测序鉴定突变位点的随机性和多样性 ,原核表达该变异体库 ,体外测定生物学活性。成功获得rhLT三点随机点突变组合文库 ,其转化克隆数达到 1 5× 10 5,是多样性理论值的 4 5倍。 5 0个样品的序列分析显示各个位点的核苷酸和氨基酸序列的突变都呈随机性分布。对原核表达的 30个样品进行生物学活性测定 ,结果 70 % (2 1个 )的样品无活性、2 3 3% (7个 )的样品活性低于rhLT、6 7% (2个 )的样品活性高于rhLT。成功构建了rhLT随机点突变组合文库 ,该库不仅在一级结构上具有良好的随机性和多样性 ,而且具有生物学活性的多样性 ,为应用噬菌体展示等高通量筛选策略对淋巴毒素进行体外分子进化和结构与功能的深入研究打下了基础。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 DH5α.The biological activity of some of the mutants was tested in L929 mouse fibroblast cells. As much as 1.5×10 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.

关 键 词：淋巴毒素 随机突变 分子进化 点突变 组合文库 

分 类 号：Q785[生物学—分子生物学]