《病毒学》第9章研究方法.ppt

? George P. Smith –Alternative vectors 1984,1985 –Mapping epitopes/peptides library 1988 –Monoclonal antibody/antibody library 1988.10 –Drugs/peptide ligands for receptors 1988.12 ?Filamentous Fusion Phage: Novel Expression Vectors That Display Cloned Antigens on the Virion Surface –Science, 1985, 228:1315 Phage display, an expression cloning method in which foreign genes are inserted into a phage vector and are expressed to give polypeptides that are displayed on the surface (protein coat, capside) of the phage. Definition Genetic engineering techniques are used to insert foreign DNA fragments into a suitable phage coat protein gene. The modified gene can then be expressed as a fusion protein which is incorporated into the virion and displayed on the surface of the phage which, however, retains infectivity (fusion phage). If an antibody is available for a specific protein, phage displaying that protein can be selected by preferential(优先的) binding to the antibody: affinity purification of virions bearing a target determinant can be achieved from a 108-fold excess of phage not bearing the determinant, using even minute quantities of the relevant antibody. Principle of phage display By cloning large numbers of DNA sequences into the phage, display libraries are produced with many billions of unique displayed proteins. 四、病毒的鉴定 Using a process of biopanning（生物淘洗）, one can rescue phage that display a protein that specifically binds to a target of interest. Briefly, a simple method for biopanning involves: coating a plate with the target incubating the library on the plate to allow phage displaying a complementary protein to the target to bind Non-binding phage are then washed away and those that are bound are eluted（洗提，洗脱，淘洗）. Infection of bacteria with the binding phage results in phage amplification. Successive rounds of biopanning enrich the pool of phage, with clones that specifically bind the target. Phage display has many applications Several usefu