In vitro selection and characterisation of human anti-HIV-1 antibody fragments

Tang, Jie (2011) In vitro selection and characterisation of human anti-HIV-1 antibody fragments. Doctoral thesis, University of Westminster.

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Abstract

Generation of neutralising antibodies with broad specificity would be one of the effective approaches to control HIV-1 spread. It is clear that a method that allows rapid generation of neutralising antibodies is needed. This project aims at developing a novel approach to rapidly access human anti-HIV-1 antibodies in vitro by using ribosome display and selection from DNA libraries of HIV-1 patients. Two single-chain antibody libraries (M325 and K530) were constructed from two HIV-1 long-term non-progressors, whose sera showed cross-neutralising activities against various HIV-1 strains across a range of clades. In each library, total RNA was extracted from blood of each donor and used to synthesise cDNA. Families of 4 ? light chains, 9 ? light chains and 8 heavy chains were generated by using RT-PCR amplification. These fragments were then assembled with all possible combinatorial pairs to form diversified repertories in the form of VL-link-VH-partial CH. Both libraries were subjected to ribosome display for in vitro selection of functional antibodies. Ribosome display is a cell-free technique used to generate proteins that can bind to an immobilised antigen. During this process, the translated proteins are associated with their mRNAs, enabling a simultaneous selection of functional proteins and their gene. The employment of ribosome display facilitated rapid screening of two large libraries against recombinant gp120 (generated from patient K530).

Item Type: Thesis (Doctoral)
Subjects: University of Westminster > Science and Technology > Life Sciences, School of (No longer in use)
Depositing User: Miss Nina Watts
Date Deposited: 31 Aug 2011 10:55
Last Modified: 31 Aug 2011 10:55
URI: http://westminsterresearch.wmin.ac.uk/id/eprint/9640

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