基因直接进化研究进展 —— DNA Shuffling 技术讲解.ppt

枯草杆菌蛋白酶E热稳定性的分子进化（Huimin Zhao等，1999） 进化后的枯草杆菌蛋白酶E 正面 反面 耐热p-硝基苯酯酶的分子进化(Lori Giver等，1998） β-葡糖苷酶耐热性的提高 (Mary′a Jesu′ s Arrizubieta等，2000） Evolution of a cytokine using DNA family shuffling Chia-Chun J. Chang ……. Phillip Patten Nature Biotechnology Vol. 17, August, 1999 Aims of the work: Demonstration of usefulness of the DNA shuffling tool for rapidly evolving human a interferon towards improving antiviral property Interferons (IFNa, IFNb, IFNg) Reasons for their limited use as drugs: IFNas are produced by monocyte, macrophages and other cells upon stimulation by virus. IFNs are able to protect cells from viral infection by reacting with IFN receptors on the cell which triggers a series of signal transduction events, hence immune response. IFNs suppress an exaggerated growth potential of cancer cells. IFNs can be used as drugs in the treatment of viral infection. Only one natural IFN-a, Hu-IFN-a2, in clinical studies. Most active engineered IFN-a, alfacon-1, is a consensus of 13 wild type Hu-IFN-a genes that is used in hepatitis C therapy. Dose-limiting toxicity, Receptor cross-reactivity, Short serum half-lives Methods and Materials-1 DNA Shuffling Construction of 1st round library: 8 cloned Hu-IFN genes were shuffled and assembled insert cloned in the phagemid display vector Construction of 2nd round 5 libraries: IFN-CH1. 1xIFN-CH1. 2; IFN-CH1. 1x IFN-CH1. 3; IFN-CH1. 1xIFN-CH1. 4; IFN-CH1. 2x IFN-CH1. 3; IFN-CH1. 1xIFN-CH1. 2x IFN-CH1. 3x IFN-CH1. 4 (mating of 4 genes). Pair-wise matings Phagemid display of IFN High-throughput phagemid preps (Q-BOT colony picker) Antiviral assays: Cytopathic effect reduction assay on mouse L929 cells, challenging virus-EMCV, 96 well plate format, neutral red, cell fixing, colour extracting, spec OD 540 vs IFNa concentration. phagemids E.coli Expression induced phagemids aggregated PEG precip CsCl banded Library intro- into E.coli Thousands of