Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.
Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.
Gupta
S
S
Department of Biochemistry, University of Delhi South Campus, New Delhi, India.
Arora
K
K
Sampath
A
A
Singh
S S
SS
Gupta
A
A
Chaudhary
V K
VK
D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D015723:Gene Library; D016655:HIV Core Protein p24 / Q000235:genetics / Q000276:immunology*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D016056:Immunodominant Epitopes / Q000032:analysis*; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D017931:DNA Primers; D004279:DNA, Viral; D016655:HIV Core Protein p24; D016056:Immunodominant Epitopes; D019151:Peptide Library; D011993:Recombinant Fusion Proteins