| Abstract [eng] |
Tumor necrosis factor (TNF) is a cytokine involved in systemic inflammation. Tumor necrosis factor promotes the inflammatory response, which, in turn, causes many of the clinical problems associated with autoimmune disorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and refractory asthma. These disorders could be treated using a TNF inhibitors. Antibodies against TNF proved to be the most perspective instrument for therapy of increased level of TNF.. Recombinant single chain antibodies (scFv) might be excellent alternative to expensive and complicated monoclonal antibodies. The scFv are recombinant antibody fragments consisting of only two domains: the variable light chain (VL) and variable heavy chain (VH) domains, covalently connected to one another by polypeptide linker. The benefits of scFv over traditional monoclonal antibodies shoul be the small penetrable size and rapid, inexpensive production. In addition, the lack of Fc domains makes them less immunogenic and less capable for binding to Fc receptors distributed on the normal cells. In this work we tried to construct and express scFv antibodies against TNF. Firstly, for this purpose we purified total RNA from hybridoma cells, producing monoclonal anti-TNF antibodies. Then we synthesised cDNA and isolated PCR fragments of variable heavy and light chain domains of monoclonal antibodies against TNF. In order to get single chain antibodies, we coupled domains by a flexible peptide linker. We cloned new gene of single chain antibody into E. coli expression vectors pET21(+)b and pET22(+)b. and managed to get overexpression of scFv protein in E. coli cells though it was aggregated. We tried to express anti-TNF single chain antibody in Saccharomyces cerivisiae. For this purpose we constructed yeast expression plasmid designed for protein secretion to growth media. Though expression was found detectable in WB, the scFv protein was also insoluble and aggregated. |
