1.1.1 The aim of the reasearch Phage display has been recently introduced as a means of making antibodies in vitro(Barbas, Kang et al. 1991; Griffiths, Malmqvist et al. 1993; Griffiths, Williams et al. 1994; Vaughan, Williams et al. 1996; Sheets, Amersdorfer et al. 1998). In general, the affinity of the antibodies isolated is pfoportional to the initial size of the library used for selection. This is based on both theoretical (Perelson and Oster 1979) and practical (Vaughan, Williams et al. 1996) considerations. In view of this, the creation of larger and larger libraries has bec01ne an important goal in the use of this technology in the selection of antibodies against any antigen. The aim of my research were: - the design and the validation of a new phagemid vector for phage display of antibody fragments (scFvs). - The design of a new strategy for "one vector" in viva recombination. - The construction of a large naive scFv libray 1.1.2 Results obtained The following chapters will describe all the steps taken in order to achieve the final goal: the construction of a large phage antibody library. First of all there will be the description of the design of a new phagemid vector in which all the possible variables were considered from a theoretical point of view, and the best elements were chosen for use. Secondly data confirming the quality of the vector will be pre_sent.ed. __ This was achieved first with the cloning of several mAb and then with the construction of two small immune libraries that allow the selection of several specific and functional scFv. The third part will describe a method which uses a single vector to exploit the reversibility of ere catalysed recombination. This method involves the creation of a relatively small primary library (7xl o7 was used here) in a phagemid vector in which the VH and VL genes are separated by two nonhomologous lox sites. The heavy and light chain genes in this primary library are then recombined by infecting the phagemid particles into ere expressing bacteria at high multiplicity of infection (MOI). Under these conditions many different phagemid particles enter a single bacteria and the VH and VL genes are exchanged between different phagemids, creating many new VH/VL combinations, all of which are functional. This ends with the production of a large naive library of preaviously unattainable size that was validated by the selection of antibodies, with high affinity, against a large number of different protein antigens. In order to fully appreciate the different aspects of experimental works and its underlying strategies some theoretical paragraphs are included in this first chapter.
|Titolo:||The Development of a Single Vector Recombination System to Make Large Phage Antibody Libraries|
|Data di pubblicazione:||8-nov-1999|
|Appare nelle tipologie:||8.1 PhD thesis|