| Abstract [eng] |
Izabelė Makūnaitė‘s Master‘s thesis is titled “Construction of a Single Chain Variable Fragment Antibody“; Supervisor of the research is dr. Milda Plečkaitytė; Vilnius University; Faculty of Medicine; Institute of Biomedical Sciences (Pharmacy and Pharmacology Center), Vilnius, 2024. Aim of the thesis: to construct a single-chain antibody fragment against the ACT-14 β-lactamase using a virus-like particle platform. Task of the research: to clone immunoglobulin the heavy (VH) and light (VL) chain variable regions from a hybridoma against the ACT-14 β-lactamase; to join the variable regions of the light and heavy chains with a peptide linker, to create a genetic construction encoding a recombinant single-chain antibody fragment (scFv); to clone the scFv into thr yeast expression plasmid by the fusion with the Fc domain of the human immunoglobulin and the hamster polyomavirus protein VP2; to obtain the synthesis of the fusion recombinant protein VP2-Fc-scFv in yeast cells. The object and methods of the research: A single-chain antibody fragment against the bacterial ACT-14 β-lactamase was constructed using recombinant DNA technology. Gene amplification and other cloning procedures were performed using the polymerase chain reaction. DNA fragments and polymerase chain reaction products were checked by performing electrophoresis on agarose gels. Recombinant plasmids were transformed into E.coli cells and yeast cells. Assessment and analysis of the recombinant protein synthesis was carried out using polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) and the Western blot method. The results and conclusions of the research: RNA was isolated from hybridoma cells that synthesized monoclonal antibodies against ACT-14 β-lactamase was used for amplification of genes encoding the variable regions of the light (VL) and heavy (VH) chains. Gene amplification was performed by polymerase chain reaction (PCR) from cDNA using primers complementary to DNA sequences. The VL and VH fragments were joined by PCR using a linker (L). Genetic VL-L-VH construction encoding the recombinant scFv antibody fragment against ACT-14 β-lactamase was created and cloned into a yeast expression vector fused with IgG Fc domain and hamster polyomavirus capsid protein VP2. Recombinant proteins VP1 and VP2-Fc-scFv synthesized in yeast cells were detected by their molecular weight through performing SDS-PAGE electrophoresis. The constructed single-chain antibody fragment against ACT-14 β-lactamase was identified by the Western blot method using polyclonal Rabbit Anti-Human IgG. |
