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An effi cient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplifi cation.

Phage display library technology is a common method to produce human antibodies. In this technique, the
immunoglobulin variable regions are displayed in a bacteriophage in a way that each fi lamentous virus displays the product of a single antibody gene on its surface. From the collection of diff erent phages, it is possible to isolate the virus that recognizes specifi c targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene.
In this work, we describe a simple and effi cient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplifi cation to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be effi cient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.   Ver Pdf

 

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