1,721,153 research outputs found
The Development of a Single Vector Recombination System to Make Large Phage Antibody Libraries
No full text1.1.1 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
Method for the preparation by in vivo recombination of nucleic acid and polypeptide libraries and uses thereof
No full text
A definitive set of oligonucleotide primers for amplifying human V regions
No full textThe creation of large diverse phage antibody libraries from natural sources relies on primers which are able to amplify as many V genes as possible. All functional germline V genes have recently been catalogued in a database, V BASE [1]. Previously published primer sets are unable to recognise all these V genes
Exploiting recombination in single bacteria to make large phage antibody libraries
No full textThe creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Cre recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of Vh and Vl genes between such phagemids creates many new V h/Vl combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3x1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigen
Using archaeal histones for precise DNA fragmentation , 20: 267-271 (2007)
No full textThe fragmentation of DNA is a useful procedure for
many molecular biology procedures. However, most
methods used to fragment DNA are poorly controllable,
and cannot be used to create small fragments. We
describe a method to generate random DNA fragments of
a predictable size to be cloned in expression vectors
for the construction of display libraries. The DNA is
allowed to form complexes with archaeal histones from
Methanothermus fervidus (HMf) and the HMf/DNA core
complex is naturally protected from nuclease DNaseI
activity, giving rise to DNA fragments of 60 bp and
multiples thereof. We found that by varying the wt/wt
ratio between DNA and HMf, the concentration of DNA
and the incubation time with DNaseI, DNA fragments of
desired size can be obtained. This approach should be
applicable to the efficient fragmentation of DNA for the
construction of phage display polypeptide libraries, as
well as any other molecular biology procedures in which
small DNA fragments of defined size are required
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