Amplificação de repertório gênico de VHH de lama glama com vistas à construção de biblioteca naive de anticorpos de domínio único

Abstract

Animals of the Camelidae family naturally produce functional G immunoglobulin lacking light chains, called heavy chain antibodies. The antigen recognition region of these antibodies is formed by the single domain, known as VHH or nanobody. VHHs are molecules of about 15 kDa, with low immunogenicity, high stability to pH and temperature changes and can be produced in microbial systems. Thus, they are presented as a promising tool for biotechnological applications in the areas of therapeutics, diagnostics and scientific research. Due to the wide applicability of this tool, this work aimed to amplify the VHH gene repertoire of one animal of the Lama glama species that may be used to build a naive library of camelid nanobodies. After collecting the blood sample from the animal, peripheral lymphocytes were isolated and quantified. These cells were used to extract total RNA, which was subsequently used for cDNA synthesis. Then, synthesized material was submitted to two sequential PCRs using specific oligonucleotide primers. In the first reaction (PCR I), gene fragments related to VH-CH1-CH2 regions, present in conventional antibodies, and VHH-CH2, present in heavy chain antibodies, were

amplified. PCR I product was submitted to agarose gel electrophoresis and the fragment corresponding to VHH-CH2 region (600 bp) was excised from the gel, purified and subjected to PCR II, where VHH gene repertoire was amplified (about 400 bp) and Sfi-I and Not-I restriction enzyme sites were inserted. Amplified sequences represent the variability of nanobodies of an animal and can be used later for the construction of a library of VHH with the perspective of nanobody selection against relevant antigens for public health.