We describe the construction and characterization of a hepatitis C virus (HCV) cDNA expression library displayed as a fusion to the carboxy terminus of the capsid protein D of bacteriophage lambda, cDNA inserts were obtained by tagged random-priming of the HCV genome and cloned into a lambda vector from which chimeric phage bearing both wild-type D protein and D fusion products on the capsid surface were produced. The resulting library was affinity-selected with anti-HCV human monoclonal antibodies recognizing linear or conformational epitopes, and human sera from HCV-infected patients. Selection was monitored by immune-screening experiments, ELISA, and sequence analysis of positive clones. The performance of this Library was compared with two additional HCV cDNA display libraries generated as N-terminal fusions to the III and WI capsid proteins of filamentous phage M13. The results obtained demonstrate the great potential of the lambda display system for constructing complex cDNA libraries for natural ligand discovery. (C) 1998 Academic Press.
Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda / C. Santini;D. Brennan;C. Mennuni;R. H. Hoess;A. Nicosia;R. Cortese;A. Luzzago. - In: JOURNAL OF MOLECULAR BIOLOGY. - ISSN 0022-2836. - STAMPA. - 282:(1998), pp. 125-135. [10.1006/jmbi.1998.1986]
Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda
NICOSIA, Alfredo;
1998
Abstract
We describe the construction and characterization of a hepatitis C virus (HCV) cDNA expression library displayed as a fusion to the carboxy terminus of the capsid protein D of bacteriophage lambda, cDNA inserts were obtained by tagged random-priming of the HCV genome and cloned into a lambda vector from which chimeric phage bearing both wild-type D protein and D fusion products on the capsid surface were produced. The resulting library was affinity-selected with anti-HCV human monoclonal antibodies recognizing linear or conformational epitopes, and human sera from HCV-infected patients. Selection was monitored by immune-screening experiments, ELISA, and sequence analysis of positive clones. The performance of this Library was compared with two additional HCV cDNA display libraries generated as N-terminal fusions to the III and WI capsid proteins of filamentous phage M13. The results obtained demonstrate the great potential of the lambda display system for constructing complex cDNA libraries for natural ligand discovery. (C) 1998 Academic Press.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.